CN1829736A - The severe acute respiratory syndrome coronavirus - Google Patents

Abstract

An outbreak of a virulent respiratory virus, now known as Severe Acute Respiratory Syndrome (SARS), was identified in Hong Kong, China and a growing number of countries around the world in 2003. The invention relates to nucleic acids and proteins from the SARS coronavirus. These nucleic acids and proteins can be used in the preparation and manufacture of vaccine formulations, diagnostic reagents, kits, etc. The invention also provides methods for treating SARS by administering small molecule antiviral compounds, as well as methods of identifying potent small molecules for the treatment of SARS.

Description

The All Files that severe acute respiratory syndrome coronavirus this paper quotes fits into this paper as a reference in it.

Require the related application of priority

Below application is included in this paper as a reference in full: U.S. Provisional Patent Application 60/462,218, and application attorney docket PP20474.001 submitted to express mail by American Post Office on April 10th, 2003; U.S. Provisional Patent Application 60/462,465, application attorney docket PP20480.001 submitted to express mail by American Post Office on April 11st, 2003; U.S. Provisional Patent Application 60/462,418, application attorney docket PP20480.002 submitted to express mail by American Post Office on April 12nd, 2003; U.S. Provisional Patent Application 60/462,748, application attorney docket PP20480.003 submitted to express mail by American Post Office on April 13rd, 2003; U.S. Provisional Patent Application 60/463,109, application attorney docket PP20480.004 submitted to express mail by American Post Office on April 14th, 2003; U.S. Provisional Patent Application 60/463,460, application attorney docket PP20480.005 submitted to express mail by American Post Office on April 15th, 2003; U.S. Provisional Patent Application 60/463,668, application attorney docket PP20480.006 submitted to express mail by American Post Office on April 16th, 2003; U.S. Provisional Patent Application 60/463,983, application attorney docket PP20480.007 submitted to express mail by American Post Office on April 17th, 2003; U.S. Provisional Patent Application 60/463,971, application attorney docket PP20480.008 submitted to express mail by American Post Office on April 18th, 2003; U.S. Provisional Patent Application 60/464,899, application attorney docket PP20480.009 submitted to express mail by American Post Office on April 22nd, 2003; U.S. Provisional Patent Application 60/464,838, application attorney docket PP20507.001 submitted to express mail by American Post Office on April 22nd, 2003; U.S. Provisional Patent Application 60/465,273, application attorney docket PP20518.001 submitted to express mail by American Post Office on April 23rd, 2003; U.S. Provisional Patent Application 60/465,535, application attorney docket PP20518.002 submitted to express mail by American Post Office on April 24th, 2003; U.S. Provisional Patent Application 60/468,312, application attorney docket PP20480.010 submitted to express mail by American Post Office on May 5th, 2003; U.S. Provisional Patent Application 60/473,144, application attorney docket PP20480.011, on May 22nd, 2003, U.S. Provisional Patent Application 60/495,024, application attorney docket PP20480.012 submitted to express mail by American Post Office on August 14th, 2003; U.S. Provisional Patent Application 60/505,652, application attorney docket PP20480.013 submitted to express mail by American Post Office on September 23rd, 2003; U.S. Provisional Patent Application 60/510,781, application attorney docket PP20480.014 submitted to express mail by American Post Office on October 11st, 2003; U.S. Provisional Patent Application 60/529,464, application attorney docket PP20480.015 submitted to express mail by American Post Office on December 11st, 2003; U.S. Provisional Patent Application 60/536,177, application attorney docket PP20480.016 submitted to express mail by American Post Office on January 12nd, 2004; With U.S. Provisional Patent Application 60/_, _, application attorney docket PP20480.017 submitted to express mail by American Post Office on April 7th, 2004.

Invention field

The present invention relates to nucleic acid and the protein of severe acute respiratory syndrome (SARS) virus. These nucleic acid and protein can be used for preparing and making to treat or prevent the bacterin preparation of SARS. The present invention also relates to diagnostic reagent, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in have or do not exist the method for SARS virus. The invention still further relates to the method for the treatment of or preventing SARS with the medicine box of the combination of little molecule viral inhibitors and little molecule viral inhibitors and treatment SARS.

Background of invention

The outburst of strong Respirovirus is called as severe acute respiratory syndrome (SARS) now, in 2003 in Hong Kong, China and world many countries be identified. Patient's symptom generally includes heating, dry cough, expiratory dyspnea, headache and hypoxemia. The separator of SARS virus shows at least the RNA polymer homology with several known coronavirus. The Phylogenetic Analysis of this homology is seen Peiris etc., " coronavirus is the possible cause of disease of severe acute respiratory syndrome " (Coronavirus as a possible cause of severe acute respiratory syndrome), Lancet, be published in http on April 8th, 2003: ///image.thelavcet.com/extras/ 03art3477web.pdf, fit into this paper in it as a reference. Genomic other of SARS virus checked order, and fragment shows and the ORF 1b of coronavirus is overlapping. Referring to Drosten etc., " identify the novel coronavirus in the severe acute respiratory syndrome patient " (Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome), New England Journal of Medicine, on April 10th, 2003 was published in http://www.nejm.org, fitted into this paper in it as a reference.

(http://www.bcgsc.ca/bioinifo/SARS/) announced a kind of TOR of being called in its website to be positioned at the genome scientific center (Genome Science Center) of Canadian British Columbia₂Separator it is believed that the genome assembling sketch of 29738 base-pair viruses that are SARS virus. The SEQ ID NO:1 of this paper has provided this genome assembling sketch.

Center for Disease Control (CDC) is in its website

(http.www.cdc.gov/ncidod/sars/pdf/nucleoseq.pdf) announced the nucleotide sequence (SEQ ID NO:2) of SARS-CoV strain on. CDC has also announced N, the S of prediction and the phylogenetic tree (seeing Fig. 6) of M albumen. This phylogenetic tree places SARS virus outside the coronavirus type of any previously known.

Day by day need the preventative or therapeutic vaccine of anti-SARS virus, and identify virus exists in mammalian tissues for example or the serum diagnosis, screening technique and composition.

Summary of the invention

The present invention relates to nucleic acid and the protein of severe acute respiratory syndrome (SARS) virus. These nucleic acid and protein can be used for preparing and making the bacterin preparation for the treatment of or prevention SARS. This bacterin preparation can comprise the SARS virus goods of deactivation (or killed) SARS virus, attenuation SARS virus, separation and one or more SARS virus Antigen Subunit preparations of restructuring or purifying. Can help by viral vectors and/or virion expression and the conveying of polynucleotides of the present invention.

The invention still further relates to the diagnosticum, kit (comprising this reagent) and the method that there are or do not exist SARS virus for diagnosis or identification of organism sample. The present invention also comprises the polynucleotide sequence of non-coding SARS virus, the SARS virus sequence of coding non-immunogenic protein, is used for conservative and SARS virus polynucleotide sequence variation of this diagnosis composition and method.

The invention still further relates to the bacterin preparation that contains one or more SARS virus antigens and one or more other Respirovirus antigens. Being applicable to other Respirovirus antigen of the present invention comprises: influenza virus, ERC group virus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus(RSV) (RSV), adenovirus, super Pneumovirinae (metapneumovirus) and rhinoviral antigen. Other Respirovirus antigen is coronavirus outside the sars coronavirus also. Preferred other Respirovirus antigen is influenza antigen.

Composition of the present invention also comprises one or more adjuvants. Be suitable for adjuvant of the present invention and comprise the outer adjuvant of mucosal adjuvants, transdermal adjuvant or enteron aisle. Be applicable to the bacterium ADP-ribosylation toxin that mucosal adjuvants of the present invention comprises detoxification, such as Escherichia coli thermally labile toxoid (for example, LTK63), chitosan and derivative thereof, and the anatoxic nontoxic double mutant form of Bordetella pertussis (Bordetella pertussis). Be applicable to the outer adjuvant of enteron aisle of the present invention and comprise MF59 and aluminium or aluminium salt.

The present invention also provides the method for the treatment of SARS by giving micromolecular compound, and identifies the effectively micromolecular method for the treatment of SARS.

In one aspect of the invention, provide a kind of method of identifying the therapeutic activity agent, the method comprises: the cell that (a) makes therapeutic activity agent contact infection SARS virus; (b) measure the reduced activity of the enzyme relevant with SARS.

One than specific embodiment in, described therapeutic activity agent is little molecule. In another more particular embodiment, described therapeutic activity agent is nucleoside analog. In another more particular embodiment, described therapeutic activity agent is class peptide, oligopeptides or polypeptide. In another embodiment, the described enzyme relevant with SARS is SARS protease. In another embodiment, the described enzyme relevant with SARS is the SARS polymerase. Again in another embodiment, the described enzyme relevant with SARS is kinases. Following V chapters and sections have further been discussed the method for identifying the therapeutic activity agent that is used for the treatment of the SARS virus infection.

In another aspect of this invention, the method that provides treatment to infect the people of SARS comprises a kind of little molecule of the patient that these needs are arranged. In one embodiment, described little molecule is the SARS protease inhibitors. In another embodiment, described little molecule is the SARS AG14361. In another embodiment, the described enzyme relevant with SARS is kinases. Again in another embodiment, described little molecule per os or enteron aisle external administration.

The present invention also provides this little molecule to be used for the treatment of application in the medicine of severe acute respiratory syndrome in manufacturing.

Micromolecular compound of the present invention comprises those less than the molecule of 1000g/mol, preferably contains armaticity part and is selected from the hetero atom of O, S or N more than one.

Preferred little molecule includes but not limited to: acyclovir, gancyclovir, vidarabidine, foscamet, cidofovir, amantidine, Ribavirin, trifluorothymidine, Zidovudine, Didanosine, zalcitabine and their combination. Also available interference is usually treated the patient, comprises interferon-' alpha ' and interferon-beta. Interferon therapy has shown in monkey wishes treatment SARS (Enserink (2004) Science 303:1273-1275), during especially by PEGization (Haagmans etc. (2004) Nature Medicine 10:290-293).

One aspect of the present invention relates to the individuality of identifying contacted SARS virus (SARSV) and contains the method for the biological sample of SARSV, and implements the kit of this method. This method can be used nucleic acid detection technique, as amplification (TMA), ligase chain reaction (LCR), the branched DNA signal amplification of PCR, RT-PCR (coronaviridae be RNA virus), transcriptive intermediate measure, based on the isothermal duplication (NASBA) of nucleotide sequence, other self continues the combination of sequence replicating mensuration, self-turning-back DNA cloning, strand displacement activation, circle probe technology or these amplification methods. As known in the art, these nucleic acid detection techniques have adopted the oligonucleotides that contains with the similar or complementary nucleotide sequence of SARS virus genome as primer (as being used for amplification) and probe (for example for catching or detecting).

Perhaps, or except above-mentioned nucleic acid detection method, method of the present invention can utilize various immunoassaies to detect SARSV antigen and/or antibody.

Therefore, the present invention relates to detect by the antibody with specific binding SARSV antigen the situation that exists of SARSV antigen, thereby identify the individual of contacted SARSV or contain the method for the biological sample of SARSV. The preferred monoclonal antibody of described antibody. The viral antigen amount that exists in the individual sample can be used to determine the prognosis of infected individual. Preferred normally a kind of structural proteins of SARSV antigen to be detected, especially those are present in the albumen on virion surface, for example comprise furcella (spike) glycoprotein (S), are also referred to as E2; Coating (membranelle) albumen (E) is also referred to as sM; Membrane glycoprotein (M) is also referred to as E1; Hemagglutinin-esterase glycoprotein (HE); Be also referred to as E3; And nucleocapsid phosphoprotein (N). In preferred embodiments, antigen to be detected is S, E and M albumen, can detect with their antibody.

The present invention relates to identify used reagent in the kit of individual SARSV and this kit. First container and the second container that contains SARSV antigen of the antibody that contains specific binding SARSV antigen are housed in the described kit. The preferred monoclonal antibody of described antibody. This kit can be used for the amount of antigen in the quantitative assay individual sample. This Information Availability is in the prognosis of determining infected individual.

The present invention relates to by the individuality of whether identifying contacted SARS virus with the existence of the antibody of anti-SARS virus antigen in the SARS antigen test sample or the method that contains the biological sample of SARSV. The amount that the anti-SARS protein that exists in the quantitative assay individual sample is SARS antibody can be used to determine the prognosis of infected individual. Available one or more virus proteins (structural proteins or non-structural protein) detect SARSV antibody as antigen; SARSV antigen conservative in the SARSV separator is preferred. Thus, non-structural protein (for example, Pol, Hel, 3CLp, MP, PLP1, PLP2) may be particularly useful.

The present invention relates to identify kit and the used reagent wherein of the individuality of contacted SARS. The first container and the second container that contains SARS antigen at the antibody of replying middle generation of contacted SARS virus antigen are housed in the described kit. The amount of the anti-SARS antibody that exists in the suitable quantitative assay individual sample of this kit. This Information Availability is in the prognosis of determining infected individual.

The present invention relates to identify the individual of contacted SARS virus or contain the method for the biological sample of SARSV by the existence that detects SARS virus nucleic acid. The amount of the SARS nucleic acid that exists in the quantitative assay individual sample can be used for determining the prognosis of infected individual. The method adopts with SARSV genome or transcript or copies the product sequence similarity or oligonucleotide probe and/or the primer of complementation. Preferred probe and primer have been described herein. The present invention also comprises the kit that carries out SARSV detection of nucleic acids method therefor.

The present invention also comprises the method by at least a SARS for the treatment of and/or preventing in those antiviral compounds described in the table 1 for the treatment of effective dose and the listed United States Patent (USP) of table 2 and the disclosed international patent application. In an embodiment of the method, described antiviral compound is little molecule. In another embodiment, described antiviral compound is protease inhibitors. In another embodiment, described antiviral protease inhibitors is 3C-sample protease inhibitors and/or papain-sample protease inhibitors. In another embodiment, described antiviral compound is the RNA-directed RNA polymerase inhibitor. In another embodiment, the first antiviral compound is protease inhibitors, and it is with the second antiviral compound-RNA-directed RNA polymerase inhibitor-give. The present invention also provides use in conjunction SAID and at least a antiviral compound, for example the antiviral compound described in the listed document of table 1 and table 2.

The present invention also provides the method for the treatment of table 1 and at least a SARS for the treatment of and/or preventing in those antiviral compounds described in the listed United States Patent (USP) of table 2 and the disclosed international patent application of effective dose by suction. In an embodiment of the method, described antiviral compound is little molecule. In another embodiment, described antiviral compound is protease inhibitors. In another embodiment, described antiviral protease inhibitors is 3C-sample protease inhibitors and/or papain-sample protease inhibitors. In another embodiment, described antiviral compound is the inhibitor of RNA-directed RNA polymerase. In another embodiment, the first antiviral compound is protease inhibitors, and it is with the second antiviral compound-RNA-directed RNA polymerase inhibitor-give. The present invention also provides to unite by suction and gives SAID and at least a antiviral compound, for example the antiviral compound described in the listed document of table 1 and table 2. Described SAID can use to realize partial result by suction, perhaps can use to realize systemic Absorption by for example oral or intravenous route.

The present invention also provides above-mentioned antiviral compound to be used for the treatment of application in the medicine of severe acute respiratory syndrome in manufacturing.

The present invention also provides the medicine box that is used for the treatment of and/or prevents SARS for the consumer. This medicine box is equipped with: the pharmaceutical composition that (a) contains at least a and pharmaceutically acceptable carrier in the table 1 for the treatment of effective dose and those antiviral compounds described in the listed United States Patent (USP) of table 2 and the disclosed international patent application or diluent; (b) container of described pharmaceutical composition is housed; And, randomly; (c) specification that treats and/or prevents the method for SARS with this pharmaceutical composition is described. This medicine box can be chosen the antiviral compound that multiple treatment SARS is housed wantonly, and wherein, described antiviral compound is selected from 3C-sample protease inhibitors and papain-sample protease inhibitors. In another embodiment, this medicine box is equipped with antiviral compound, and described antiviral compound is the RNA-directed RNA polymerase inhibitor. When this medicine box was equipped with multiple antiviral compound, the antiviral compound in the medicine box can be chosen wantonly and the same pharmaceutical composition.

Another aspect of the present invention provides described in table 1 and the listed United States Patent (USP) of table 2 and the disclosed international patent application at least a in the antiviral compound and has been used for the treatment of or prevents application in the medicine of SARS in manufacturing.

The accompanying drawing summary

Fig. 1: the schematic diagram of coronavirus genome structure.

Fig. 2: the schematic diagram of coronavirus ORF1a/ORF1b gene outcome.

Fig. 3 (A-C): be selected from the contrast of the coronavirus gene that comprises nucleocapsid (N), matrix (M) and hemagglutinin-esterase (HE) gene.

Fig. 4 (A-F): the coronavirus polypeptide sequence (comprises the contrast of ORF1a/ORF1b, nucleocapsid (NP), hemagglutinin-esterase (HE), coating (Sm or E), matrix (M) and furcella (S).

Fig. 5: take from furcella (S) the peptide sequence S1 of Fig. 4 and the contrast in S2 domain bonding pad and selected coronavirus protease cutting site.

Fig. 6: the CDC phylogenetic tree of SARS-CoV strain (Clustalx 1.82, adjacent threaded tree).

Fig. 6 A has shown the coronavirus N protein analysis result, and Fig. 6 B has shown coronavirus S analysis of protein result, and Fig. 6 C has shown coronavirus M analysis of protein result.

Fig. 7: the conserved sequence of SARS virus and characteristic sequences. Fig. 7 A-7D has shown multiple sequences alignments (CLUSTAL W 1.82) (the 7A:PEP4 spike protein of SARS virus genome structure albumen; The little memebrane protein of 7B:PEP7; 7C:PEP8 matrix glycoprotein; The 7D:PEP13 nucleocapsid protein), they have homologue in other known coronavirus of all or part. Fig. 7 E-7H has shown the dendrogram of protein distance in the sequence of contrast 7A-7D. Mark 229E: human corona virus; MEV: murine hepatitis virus; TGV: TGE; AIBV: avian infectious bronchitis virus; BOVINE: bovine coronavirus; PEDV: Porcine epidemic diarrhea virus.

Fig. 8: the contrast of several coronavirus 5 ' UTR, the total nucleotide sequence of demonstration 5 ' UTR.

Fig. 9: the sequence that is used for the preferred primer of amplification 5 ' UTR. F and R represent forward and inverse PCR primer, the position of nucleotides among numeral Fig. 8.

Figure 10: the contrast of several coronavirus 3 ' UTR, the total nucleotide sequence of demonstration 3 ' UTR.

Figure 11: the preferred primer sequence that is used for amplification 3 ' UTR. F and R represent forward and inverse PCR primer, the position of nucleotides among numeral Figure 10.

Figure 12: the coiled coil prediction of SEQ ID NO:6042, adopt Coils program (Figure 12 A) or LearrCoil (Figure 12 B).

Figure 13: at the example that inserts reporter gene interested between the existing SARS virus gene between certain site. Not shown little nonstructural gene product.

Figure 14: the schematic diagram of SARS virus replicon representative example. Not shown little nonstructural gene product.

Figure 15: SARS virus nsp2 protease (3CLp) and to the evaluation of catalytic site and substrate sites.

Figure 16: the contrast of bird IBV, MHV and BcoV SARS virus nsp2 protease (3CLp). Residue in the dotted line frame is the Key residues (F, Y and H) of substrate sites; Residue in the solid box is catalytic cysteine (C) and histidine (H) residue.

Figure 17: the genome structure of sars coronavirus. Replicase zone and the ORF1a of structural region and prediction and the cleaved products in the ORF1b have been shown. Also marked the position of 5 ' RNA targeting sequencing (L), 3 ' polyadenylic acid passage and ribosomal frameshift consensus sequence between ORF1a and the ORF1b. Each frame represents a kind of protein. Represent (also can referring to table 2) according to the amino acid homogeny level of they and other coronavirus respective egg white matter with shade. The SARS specific gene is white. With arrow marked 9 hexabasic basic IG sequences of SARS specificity (5 '-ACGAAC-3 '; SEQ ID NO 7293) position.

Figure 18: the representative group 1 (HCoV-229E of coronavirus, accession number: AF304460), the group 2 (MHV MHV, accession number: NC_001846), group 3 (avian infectious bronchitis virus AIBV, accession number: NC001451) and the genome structure of sars coronavirus. Other coronavirus of checking order fully that is used for this research is available from following accession number: Porcine epidemic diarrhea virus (PEDV), AF353511; TGE (TGV), NC002306; Bovine coronavirus (BCoV): AF220295. Red frame represents the group-specific gene. In genome, also marked the position of leading RNA sequence and polyadenylic acid passage. Marked the position of specificity IG sequence with the deep or light circle of difference. In the SARS genome, we have also found to organize 2 distinctive three the IG sequences of coronavirus.

Figure 19: the spike protein of prediction anchors to the topology model of viromembrane. Marked the functional domain of domain and prediction. Estimate that N-stub area (S1) comprises receptor binding domains. With two coiled coils zones arranged in the S2 domain of leucine zipper motif partial stack infer the oligomerization effect that participates in. Hydrophobic domains is responsible for the film grappling.

Figure 20: the 922bp interior zone of the pol gene of 12 kinds of coronavirus and SARS is carried out the phylogenetic tree that multiple sequences alignments obtains. The result that the numeral boot of node is analyzed supports branch strongly. The sequence that does not have in the complete coronavirus genome retrieves following accession number from GenBank: pigs haemagglutinating encephalomyelitis virus (PHEV), AF124988, people OC43 virus (OC43), AF124989, canine coronavirus (CCV), AF124986, feline infectious peritonitis virus (FIPV), AF124987, turkey coronavirus (TCV), AF124991, sialodacryoadenitis virus (SDAV), AF124990.

Figure 21:21A. the consensus sequence (G1_cons and G2_cons) of the spike protein S1 domain by will organizing I and group II and organize the consensus sequence of 3 spike proteins (AIBV) and the spike protein of SARS virus compares the unrooted genealogical tree of acquisition. The result that the numeral boot is analyzed. Being used for the sequence that generation organizes 1 consensus sequence feature is: HcoV-229E, accession number P15423; Porcine epidemic diarrhea virus (PEDV), accession number: NP598310; TGE (TGV), accession number: NP058424; Canine coronavirus (CCV), accession number: S41453; Porcine respiratory virus (PRV), accession number: S24284; Feline infectious peritonitis virus (FIPV), accession number: VGIH79. Being used for the sequence that generation organizes 2 consensus sequences is: MHV (MHV), accession number: NP045300; Bovine coronavirus (BCoV), accession number: NP_150077; Human corona virus OC43, accession number: P36334; Pigs haemagglutinating encephalomyelitis virus (PHEV), accession number: AAL80031; Group 3 is only used the spike protein sequence of avian infectious bronchitis virus (AIBV), accession number: AA034396.21B: expression is compared with the SARS spike protein, the schematic diagram of cysteine position in the S1 domain of group 1,2 and 3. Horizontal strip represents S1 amino acid sequence (SARS and AIBV) or consensus sequence feature (by group 1, G1_cons, and organizing 2, G2_cons generation). The length of short-term is not amplified. Relevant cysteine position represents with the rectangle band. Only have cysteine fully conservative in each consensus sequence. Demonstration is conservative between SARS S1 domain and consensus sequence with the cysteine that line connects.

Figure 22: neisseria adhesion A albumen (NadA).

Figure 23: the genomic original translation of sars coronavirus (reading frame+1).

Figure 24: the genomic original translation of sars coronavirus (reading frame+3).

The ORF of Figure 25: 1b and furcella separates with *.

Figure 26: SARS grows in the vero cell.

Figure 27: sars coronavirus is caught the chromatogram of step on the Matrix Cellufine Sulfate Superformance 150/10. 100ml coronavirus cutting is analyzed. The Y-axis on the left side represents the absorbance of 280nm. The Y-axis on the right represents gradient (%B). X-axis represents volume (ml).

Figure 28: the MCS chromatographic component that silver dyes. Each swimming lane is: (1) label; (2) coronavirus vero cell harvesting thing; Coronavirus vero cell harvesting thing after (3) 0.65 μ m filter; (4) stream is worn liquid; (5) cleaning solution; (6) 20% peaks (viral peak). Application of sample 1 μ g test protein on each swimming lane.

Figure 29: the Western trace of MCS chromatographic component. Each swimming lane as described in Figure 28.

Figure 30: the linear density gradient ultracentrifugation, 15-60% sucrose (SW28,2 hours, 20000rpm). The figure illustrates protein concentration (■) and sucrose concentration (◆).

Figure 31: the silver on the NuPage 4-12%Bis-Tris-Ge (Novex) dyes the density gradient component, reducing condition, and 70 ℃ were heated 10 minutes. Each swimming lane is: (1) label; (2) 20% peak MCS; (3) the density gradient component 11; (4) the density gradient component 12; (5) the density gradient component 13; (6) the density gradient component 14; (7) the density gradient component 15; (8) the density gradient component 16; (9) the density gradient component 17. A large amount of protein are arranged among the component 15-17. Application of sample 1 μ g protein on swimming lane 2,8 and 9.

The upper sars coronavirus of Figure 32: MCS is caught the chromatogram of step. Details such as Figure 27, but use the 200ml cutting.

Figure 33: the silver of all chromatogram components dyes (left side) and Western trace (right side) result. Each swimming lane is as described in Figure 28 and 29, but swimming lane (6) is 5% peak. Carry out before the SDS-PAGE (sample) room temperature treatment 30 minutes.

Figure 34: density gradient ultracentrifugation, 15-40% sucrose (SW28,2 hours, 20000rpm). The figure illustrates protein concentration (■) and sucrose concentration (◆).

Figure 35: the silver of NuPage 4-12%Bis-Tris-Ge (Novex) upper density gradient ultracentrifugation component dyes (left side) and Western trace (right side) result, reducing condition. Each swimming lane is: (1) label; (2) the density gradient component 6; (3) the density gradient component 7; (4) the density gradient component 8; (5) the density gradient component 9; (6) the density gradient component 10; (7) the density gradient component 15. Component 7-10 (swimming lane 3-6) contains pure coronavirus protein. In the component 15 (swimming lane 7) a large amount of impurity are arranged. Application of sample 1 μ g protein on swimming lane 2,8 and 9. Carried out before the SDS-PAGE (sample) room temperature treatment 30 minutes.

Figure 36: the EM figure of density gradient component 8-10. Figure 36 A has shown component 8; Figure 36 B has shown component 9; Figure 36 C has shown component 10.

Figure 37: furcella/NadA fusion construct.

Figure 38 and 39:S1_L、S1 _L-NadA and SlL-NadA_{The Δ anchor}Result at expression in escherichia coli. Figure 38 has shown BL21 (DE3)/pET, BL21 (DE3)/pET-S1L and BL21 (DE3)/pET-S1L-NadA_{The Δ anchor}The SDS-PAGE analysis result of full lysate. Point out each band with arrow, from left to right three swimming lanes are respectively: BL21 (DE3)/pET; BL21 (DE3)/pET-S1_L；BL21(DE3)/pET-S1 _L-NadA _{The Δ anchor} Figure 39 has shown BL21 (DE3)/pET, BL21 (DE3)/pET-S1_L-NadA (under non-inductive condition, cultivating) and BL21 (DE3)/pET-S1_L(39A) SDS-PAGE of the full lysate of-NadA (under inductive condition, cultivating) and (39B) Western engram analysis result. Point out each band with arrow, from left to right three swimming lanes are respectively: BL21 (DE3)/pET; BL21 (DE3)/pET-S1_L-NadA； BL21(DE3)/pET-S1 _L-NadA. The Western trace shows the oligomerization form that has this protein.

Figure 40: SARS furcella clone's schematic diagram.

Figure 41: the transient expression of SARS spike protein (the Western trace result of COS7 cell lysate). Each swimming lane application of sample 20 μ g cell lysate of 4-20% TG sds gel (altogether 1.2mg). Shown labelled antibody.

Figure 42: the Western engram analysis result of COS7 cell lysate on the 4%TG sds gel has shown the oligomeric state of S molecules in the born of the same parents.

Figure 43: the Western engram analysis result of COS7 cell lysate on the 4-20%TG sds gel has shown the transient expression of SARS spike protein. Each swimming lane is: (1) simulation, AF; (2) simulation, DF; (3) nSh, AF; (4) nSh, DF; (5) nSh Δ TC, AF; (6) nSh Δ TC, DF. Application of sample 5 μ 1 various samples, altogether 400 μ l on each swimming lane. Trace is with the protein labeling of anti-His-mark.

Figure 44: the Western engram analysis result of COS7 cell lysate on the 4-20%TG sds gel has shown the transient expression of SARS spike protein. Secrete the spike protein of brachymemma. Purifying spike protein from culture medium (10cm is dull and stereotyped) is at first by the ConA post, at last by His mark magnetic bead. Application of sample 1/3 material on each swimming lane.

Figure 45: the Western engram analysis result of COS7 cell lysate on the 4-20%TG sds gel has shown the transient expression of SARS spike protein. In two of the limit traces (swimming lane 1-5), sample boils in SDS and beta-mercaptoethanol leftward; In dexter two traces (band 6-11), sample only boils in SDS. Swimming lane 1-8 is with the labeling of monoclonal antibody of anti-His-labelled protein; Swimming lane 9-11 resists-the SARS antibody labeling with rabbit.

Figure 46: the SARS spike protein is expressed the impact on cell viability.

Figure 47: the Western engram analysis result of COS7 cell lysate on the 4%TG sds gel has shown the oligomeric state of born of the same parents' spina molecule. The trace mAb mark of anti--His-mark. Then the membrane component of COS7 cell lysate is loaded onto on the swimming lane by the classification of exclusion post. The band kDa value that component 7-14 shows is respectively: 71000,1400,898,572,365,232,148 and 99.

Figure 48: cell is grouped into water component and detergent component.

Figure 49: the schematic diagram that construction is used for the OMV goods.

The HR1 of Figure 50: SARS and HR₂Construction.

Figure 51: vaccine protection Balb/c mouse model exempts to suffer from SARS.

Figure 52: spike protein is in 293 cell lysates (52A) of transfection or the expression in the COS7 cell culture supernatant (52B). Protein separates with the 4-20%TG sds gel. Mark is anti--His-mark, but dexter three swimming lanes of 52B resist-the SARS serum marker with rabbit. In Figure 52 A, three swimming lanes of left-hand side are processed with DTT and are boiled, but dexter three swimming lanes are not dealt with. In Figure 52 B, do not use DTT to process, but all 80 ℃ of heating 5 minutes of all swimming lanes (sample).

Figure 53: at the Western trace of the spike protein of COS7 cells. Protein hatches to check room temperature (RT), 80 ℃ or 10 ℃ what impact molecular weight is had. Figure 54 has shown the similar result of the test that the SARS virus particle is carried out.

Figure 55: the pulse chase experiment result has shown expression and the processing of being infected rear SARS spike protein by the sub-particle of α virus replication. As shown in the figure, cell is used or is processed without Endo H.

Figure 56: heating is on the trimerical impact of spike protein.

Figure 57: the Coomassie blue stain gel of the protein of Yeast expression. Each swimming lane is: 1-visible blue standard items (10 μ l); 2-pAB24gbl (20 μ g); 3-SARS furcella S1c.1gbl (20 μ g); 4-SARS furcella S1c.2 gbl (20 μ g); 5-visible blue standard items (10 μ l); 6-pAB24ip (5 μ l); 7-SARS furcella S1c.l (5 μ l); 8-SARS furcella S1c.2 (5 μ l).

Figure 58-64: the schematic diagram of preparation Yeast expression construction.

Figure 65-66: the furcella sequence of Yeast expression.

Figure 67: show the expression of SARS spike protein in the sub-particle of α virus replication and the Western trace of replicon rna expression. Figure 67 A carries out under non-reduced condition and room temperature (namely not heating), and each swimming lane is: (1) VEE/SIN-furcella infects; (2) VEE/SIN-GFP infects; (3) replicon-furcella RNA transfection; (4) replicon-GFP RNA transfection. Figure 67 B carries out under illustrated different temperatures with the SARS virus particle.

Figure 68: in mouse, induce antibody response. Each vaccine group is: (1) deactivation of SARS virus; (2) the restructuring spike protein of brachymemma; (3) total length furcella: DNA+DNA.PLG+ α virus; (4) total length furcella: only have the α virion.

Figure 69: human monoclonal antibodies S3.2 is combined with the brachymemma spike protein of purifying. X-axis represents AC, and Y-axis represents the ELISA absorbance. Interpolation result is 2158.13.

Figure 70: different vaccines (from left to right: inactivation of viruses; The spike protein of 3 μ g brachymemmas; The ELISA titre geometrical mean of the antibody that the SARS-CoV spike protein of the DNA of the spike protein of 75 μ g coding brachymemma) carrying is induced.

Figure 71: in behind the dna immunization of the coding nSd Δ TC that carries with PLG with (left side) nSd Δ TC albumen or (right side) and titre.

Figure 72: the relation of furcella antigen binding antibody and neutralizing antibody.

Figure 73: the Western trace of expressing the Chinese hamster ovary celI system of total length form (left side) or clipped form (right side) spike protein. Protein separates by 4-12%SDS-PAGE, boils in DTT and dyes with polyclonal serum.

Figure 74: the structure of SARS-CoV spike glycoprotein and expression construction forms. L represents leader peptide (residue 1-13), and TM represents the cross-film district, and Cy represents kytoplasm periproct section. Do not show 6-His mark.

Figure 75: in the Western engram analysis result of the SARS of COS7 cells spike protein. In Figure 75 A, the plasmid construction thing transfection that marks of COS7 cell, and under reduction and Denaturing, analyze the expressing protein in the 48 hour cell lysates after the transfection with SDS-PAGE (4-20% polyacrylamide), with resist-histidine Mab shows all protein. In Figure 75 B, then the protein after transfection in 48 hours collecting cell culture mediums carry out purifying with the magnetic bead of His-mark with the ConA post first. The protein of purifying is done SDS-PAGE (4-20% polyacrylamide) analysis and is shown with anti--SARS rabbit anteserum.

Figure 76: the Endo H sensitiveness of the spike protein (SA) of the terminal brachymemma of the C-that in cell lysate (swimming lane 1,2) and culture medium (swimming lane 3,4), finds. Marked the position of the S Δ protein of S Δ protein and secretion in the born of the same parents with arrow.

Figure 77: the oligomeric state of SARS spike protein. With the recombinant s protein oligomer in total length furcella construction (nSh) rotaring redyeing COS 7 cell. Such as on each swimming lane sign, cell lysate is with DTT and/or heat treated. S albumen is multi-form by SDS-PAGE (4% polyacrylamide) and Western engram analysis in processing and the untreated samples, shows with anti--histidine Mab.

Figure 78: during without DTT, thermal denaturation is on the impact of recombinant s protein oligomeric state. Before electrophoresis, heat as indicated the COS7 cell lysate, and show S albumen according to the description of Figure 77.

Figure 79: thermal denaturation is on the impact of SARS virus particle median spine albumen oligomeric state. SARS-CoV is grown in the Vero cell,, carry out thermal denaturation and show by the description of Figure 77 by above-mentioned its purifying and dissolving from virion with SDS, but the rabbit anti-serum that uses antivenom purification virus is as probe.

Figure 80: by the oligomeric state of crosslinked analysis of experiments SARS virus particle spike protein. The SARS virus granule protein of dissolving is processed with DMS. To be untreated (-) and virion albumen that DMS processes (+) heat denatured and show by 4%PAGE and silver dyeing when not having DTT.

Figure 81 and 82: the oligomeric state of analyzing the spike protein of brachymemma by thermal denaturation. With the spike protein (81) of the brachymemma in the COS7 cell lysate or the spike protein (82) that is secreted into the brachymemma in culture medium heat denatured in accordance with the instructions when not having DTT, and show by the Western engram analysis.

Figure 83: the reactivity of deglycosylated total length furcella oligomer and conformer or non-conformation antibody. Total length restructuring furcella oligomer with the deglycosylation of PNG enzyme part, uses the rabbit anti-serum ( swimming lane 4,5,6) of the SARS CoV of anti--histidine Mab ( swimming lane 1,2,3) or antivenom purification to show by the Western engram analysis under non-Denaturing.

Figure 84: the location of the SARS spike protein of western trace Explicit Expression in each component of COS7 cell lysate. Cell with shown in plasmid transfection, after the transfection 48 hours with the Dounce homogenizer with the cracking of hypotonic buffer liquid. The centrifuge cell lysate obtains solubility kytoplasm colloidal sol component and insoluble film component, and membrane component further dissolves with 4%Triton X-100. Protein uses SDS-PAGE (4-20% polyacrylamide) to analyze under reducing condition after 80 ℃ of heating of SDS. Show protein with anti--histidine Mab. The cytosol component is loaded onto swimming lane 1,3 and 5, and membrane component is loaded onto swimming lane 2,4 and 6.

Figure 85: (B, E) spike protein of the total length of restructuring (A, D) or brachymemma in COS7 cell born of the same parents and the surface expression. 48 hours fixed cells after the transfection, with detergent (Cytofix/perm, BD Biosciences) immunofluorescence check (A, B, C) in the born of the same parents is carried out in processing, or processes (40 times of amplifications) observation of cell surface immunofluorescence (D, E, F) with 2% paraformaldehyde. The cell (C, F) that simulation transforms in contrast.

Figure 86-105: the SDS-PAGE of Bacillus coli expression protein. The Tot=total protein; The Sol=soluble proteome divides. Label is protein title (table 26-30).

Figure 106: give the immunofluorescence behind the code optimization N antigen vectors.

Figure 107: (A) natural M sequence and (B) immunofluorescence of codon optimization M sequence.

Figure 108: (A) natural E sequence and (B) immunofluorescence of codon optimization E sequence.

The Western trace of Figure 109-111:Vero cell adopts the rabbit antibody that obtains after the spike protein immunity inoculation of Bacillus coli expression.

Figure 112: 293 cell median spine protein expressions. Swimming lane: (M) label; (1) analogies of transfection; (2,6) express the cell of nS albumen, lysate; (3,7) express the cell of nSdTC albumen, lysate; (4,8) express the cell of nS albumen, supernatant; The cell of nSdTC albumen, supernatant are expressed in (5,9) (4). Dyeing antibody: (2-5) mice serum that obtains afterwards of DNA immunity; The rabbit anteserum that (6-9) obtains afterwards with the totivirus immunity of killing.

Figure 113: 6 reading frames of SEQ ID NO:9968.

Figure 114: 6 reading frames of SEQ ID NO:10033.

Figure 115: bovine coronavirus pol lab (top line; SEQ ID NO:10068), bird infective bronchitis pol lab (the second row; SEQ ID NO:10069), MHV pol lab (the third line; SEQ ID NO:10070), SEQ ID Nos:9997/9998 (fourth line) and consensus sequence (end row; SEQ ID NO:10071) contrast.

Figure 116: coronavirus genome structure schematic diagram.

Figure 117: the schematic diagram of coronavirus ORF1a/ORF1b gene outcome comprises " * " zone.

Figure 118: contrast.

Figure 119: interchangeable initiation codon among the SEQ ID NO:10080.

Figure 120: 6 reading frames of SEQ ID NO:10084.

Figure 121: the contrast of SEQ ID NO:10033 and SEQ ID NO:10084.

Figure 122: the reading frame of SEQ ID NO:10084.

Figure 123: the initiation codon analysis of SEQ ID NO:10084.

Figure 124: the BLAST of SEQ ID NO:10210 analyzes.

Figure 125: with (13A) Hopp ﹠ Woods or (13B) Kyte ﹠ Doolittle SEQ ID NO:10210 is carried out epitope analysis.

Figure 126: the reading frame of SEQ ID NO:10299.

Figure 127: the reading frame of SEQ ID NO:10505.

Figure 128: the reading frame of SEQ ID NO:11563.

Figure 129: the reading frame of SEQ ID NO:10033.

Figure 130: the contrast of SEQ ID NO:9997 and SEQ ID NO:10033.

Figure 131: the reading frame of SEQ ID NO:10299.

Figure 132: the reading frame of SEQ ID NO:10505.

Figure 133: the Western trace of SARS protease purification component.

Figure 134: with the SARS protease cutting DABCYL-EDANS (the fluorescently-labeled peptide that contains SARS protease cutting site) of variable concentrations. The relation of activity/concentration when the figure illustrates without protease (◆), 0.95uM protease (■) and 2.86uM protease (●).

When the sequence in the sequence table and the sequence among the figure when there is any discrepancy, be as the criterion with the sequence in scheming.

Detailed Description Of The Invention

When except as otherwise noted, enforcement is of the present invention conventional chemical known to the skilled, biochemistry, molecular biology, immunology and the pharmacological method in this field are proficient in employing. These technology have detailed explanation in the literature. For example can referring to, Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., the 19th edition (1995); Methods In Enzymology (S.Colowick and N.Kaplan compile, Academic Press, Inc.); And Handbook of Experimental Immunology, I-IV volume (D.M.Weir and C.C.Blackwell compile, 1986, Blackwell Scientific Publications); Sambrook etc., Molecular Cloning:A Laboratory Manual (second edition, 1989); Handbook ofSurface and Colloidal Chemistry (Birdi, K.S. compiles, CRC Press, 1997); Short Protocols in Molecular Biology, the 4th edition, (volume such as Ausubel, 1999, John Wiley ﹠ Sons); Molecular Biology Techniques:An Intensive Laboratory Course, (volume such as Ream, 1998, Academic Press); PCR (Introduction to Biotechniques Series), second edition, (Newton ﹠ Graham compiles, 1997, Springer Verlag); Peters and Dalrymple, Fields Virology (second edition), Fields etc. (volume), B.N.Raven Press, New York, NY.

Fit into this paper as a reference in all publications, patent and the patent application of quoting here.

Severe acute respiratory syndrome (SARS) virus is accredited as a kind of new viral species recently. The SARS virus kind comprises following separator.

The described two-strain separator such as-Peiris, " coronavirus is the possible cause of disease of severe acute respiratory syndrome " (Coronavirus as a possible cause of severe acute respiratory syndrome), Lancet is published in http://image.thelancet.com/extras/03art3477web.pdf online on April 8th, 2003, fit into this paper in it as a reference, its sequence is preserved in GenBank with accession number AY268070.

The described separator such as-Drosten and virus sequence, " identify the novel coronavirus in the severe acute respiratory syndrome patient " (Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome), New England Journal of Medicine is published in http://www.nejm.org online on April 10th, 2003.

On March 25th, 1 and 24 separator and the virus sequences of describing in the WHO website.

The described separator such as-Tsang and virus sequence, " a class cause of disease of Hong Kong severe acute respiratory syndrome " (A Cluster of Cases of Severe Acute Respiratory Syndrome in Hong Kong), New England Journal of Medicine, on March 31st, 2003 be published in online http. //www.nejm.org.

The described separator such as-Poutanen and virus sequence, " identify Canadian severe acute respiratory syndrome " (Identification of Severe Acute Respiratory Syndrome in Canada), NewEngland Journal of Medicine, on March 31st, 2003 be published in online http. //www.nejm.org.

As described in the article of Lanet, the 646 base-pair polynucleotides of SARS virus and the virus of coronavirus family have weak homology. The article of Lanet is also reported by the amino acid sequence (215 amino acid) of this sequence release and the sequence homology about 57% of the RNA polymerase of bovine coronavirus and murine hepatitis virus. The article of Lanet also provides the system of protein sequence to analyze, and shows that this polymerase sequence is the most closely related with the coronavirus of group II.

The virologist who is proficient in this field other SARS virus separator of can identifying, separate and/or check order. The virologist can easily identify whether SARS virus of new viral isolates. The virologist can be used to identify that the standard of new SARS separator comprises: the sequence homology of new separator and known SARS virus separator; The similarity of new virus separator and known SARS virus isolate gene group structure; Immunology (serology) similitude or homogeny with known SARS virus separator; The similitude of learning with known SARS virus separator Morphology of Virions on the pathology; And with the morphologic similitude of infected cell (for example passing through electron microscope observation) due to the known SARS virus separator.

Separate and the method for order-checking SARS virus separator comprises the method that Peiris etc. describes in the Lancet paper. The Lancet paper claims, RNA in the available random hexamer reverse transcription clinical sample, and when having the 2.5mmol/L magnesium chloride with comprising the primer amplification cDNA (94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 1min) of sequence SEQ ID NOS:6584 ﹠ 6585.

Can in the RT-PCR test, finish according to following steps with hexamer retrovirus separator at random. Make viral isolates at mammalian cell, especially breed in the fetal rhesus kidney cell. Then isolated viral infects and not by all RNA of the fetal rhesus kidney cell of virus infections. With the primer reverse transcription RNA sample that contains SEQ ID NO:6586. With the primer amplification cDNA that contains SEQ ID NO:6587. PCR product unique in the infected cell product (determining according to size) is then carried out Cloning and sequencing, the relatively genetic homogeny of sequence among this sequence and the GenBank.

Be proficient in the enough various standard clone technologies of technical staff's energy in this field, for example, utilize the overlapping sequence of the RT-PCR of random primer and detection and one or more above-mentioned sequence, and/or utilize Oligonucleolide primers (such as degenerate primer), identify and clone other genome area (seeing Fig. 1-5, Fig. 8-11, SEQ ID NOS:3-20) according to the sequence that provides above.

Utilize polynucleotide sequence, the RNA polymerase sequence of especially relevant coronavirus can further promote these those skilled in the art to clone, order-checking and the evaluation of SARS virus.

The technical staff who is proficient in this field is not difficult to determine new viral isolates and the sequence homology of above-mentioned known SARS separator. The percent homology of useful viral nucleotide sequences is identified new SARS separator, and new virus and known SARS virus polynucleotide sequence can have 99%, 95%, 92%, 90%, 85% or 80% homology. Also available peptides homologous percentage is recently identified new SARS separator, and the polypeptide of the polypeptide of new virus polynucleotide encoding and known SARS encoding viral can have 99%, 95%, 92%, 90%, 85% or 80% homology.

Also can identify new SARS separator with the percent homology of genome polynucleotide sequence, the polynucleotide sequence in the polynucleotide sequence in new virus specific gene group zone and the specific gene group zone of known SARS virus can have 99%, 95%, 92%, 90%, 85% or 80% homology. In addition, useful peptide sequence percent homology is identified new SARS separator, by the peptide sequence of the polynucleotide encoding of the peptide sequence of the polynucleotide encoding in new SARS virus specific gene group zone and known SARS virus specific regions 99%, 95%, 92%, 90%, 85% or 80% homology can be arranged. These genome areas (for example can comprise the common total zone of most of coronavirus, gene outcome), and group-specific zone (for example antigen group), any one in the following genome area for example, these zones are that the virologist who is proficient in this field is not difficult to identify: 5 ' non-translational region (UTR), targeting sequencing, ORF1a, ORF1b, non-structural protein 2 (NS2), hemagglutinin-esterase glycoprotein (HE) (being also referred to as E3), spike glycoprotein (S) (being also referred to as E2), ORF3a, ORF3b, ORF3x, non-structural protein 4 (NS4), coating (membranelle) albumen (E) (being also referred to as sM), membrane glycoprotein (M) (being also referred to as E1), ORF5a, ORF5b, nucleocapsid phosphoprotein (N), ORF7a, ORF7b, intergenic sequence, 3 ' UTR or RNA-directed RNA polymerase (pol). SARS virus can have the appraisable genome area that contains one or more said gene groups zone. SARS virus antigen comprises the protein of these any one codings of genome area. SARS virus antigen can be to SARS virus specific protein or fragment (comparing with known coronavirus). (referring to Fig. 1-5, Fig. 8-11, SEQ ID NOS:3-20).

The technical staff who is proficient in this field should also be clear that the electron micrograph of the mammalian cell that is infected by SARS virus. The electron micrograph of SARS infection cell is seen in the Lancet paper. As described in this paper, through negative staining (3% phosphotungstic acid potassium, pH 7.0) SARS infect in the electron micrograph of ultracentrifugation cell culturing extract of fetal rhesus kidney cell and show, exist diameter to be about the polymorphic tunicary virion of 80-90nm (scope is 70-130nm), its configuration of surface is consistent with coronavirus (sees the paper of Lancet, Fig. 1). The electron micrograph of infection cell slice shows that the diameter of virion is 55-90nm, and being arranged in kytoplasm has the vesicle of smooth wall (seeing the paper of Lancet, Fig. 2 B). Electron micrograph also can be used to the virion on observation of cell surface. The electron micrograph of people's lung biopsy samples has shown similar morphology of virus. See paper Fig. 2 A of Lancet.

I.SARS polypeptide and polynucleotides

The present invention relates to nucleic acid and protein from SARS virus. The below further exemplifies this polynucleotides and polypeptide.

In one embodiment, polynucleotides of the present invention do not comprise following 5 primers that disclose among the http://content.nejfn.org/cgi/reprirnt/NEJMoa030781v2.pdf: SEQ ID NOS:6034-38.

The present invention includes can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in have or do not exist the polynucleotide sequence of probe of the method for SARS virus. The present invention includes the polynucleotide sequence that contains one or more primer sequences of identifying among the SEQ ID NOS:21-1020. The present invention also comprise contain with SEQ ID NOS:21-1020 in one or more primer sequences of identifying complementary polynucleotide sequence mutually.

The polypeptide that the present invention includes contains the amino acid sequence from sequence shown in Figure 23. This amino acid sequence is SEQ ID NOS:6588-6809. The polypeptide that the present invention includes contains the amino acid sequence that has the sequence homogeny with these sequences, and the present invention also comprises the fragment of the polypeptide that contains one of these sequences.

The present invention includes the peptide sequence that contains from the amino acid sequence of sequence shown in Figure 24. This amino acid sequence is SEQ ID NOS:6810-7179. The protein that the present invention includes contains the amino acid sequence that has the sequence homogeny with these sequences, and the present invention also comprises the fragment of the protein that contains one of these sequences.

The present invention includes the protein that contains SEQ ID NO:6039. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:6039. The present invention includes the fragment of the polypeptide that contains SEQ ID NO:6039. The present invention includes diagnostic kit, the polypeptide that contains SEQ ID NO:6039 or its fragment is housed in the box. The present invention includes diagnostic kit, the polynucleotide sequence of coding SEQ ID NO:6039 or its fragment is housed in the box. The immunogenic composition that the present invention includes contains the polypeptide of SEQ ID NO:6039 or its fragment. The present invention includes the antibody that to identify the polypeptide that comprises SEQ ID NO:6039 or its fragment. SEQ ID NO:6039 is proved homology on the ORF1a function with coronavirus.

The below has identified predicted transmembrane district or the hydrophobic region of SEQ ID NO:6039. Although the polyprotein of coronavirus can be cut into many little albumen by proteolysis, the hydrophobic domains in known this polyprotein can mediate this replication complex and be combined with film, and can significantly change the structure of host cell membrane. Therefore, the hydrophobic domains of this polyprotein is the target spot of the gene mutation of exploitation attenuation SARS virus vaccine. The target spot of these hydrophobic domains or SARS virus micromolecular inhibitor. These hydrophobic domains also can be used to produce those regional specific antibodies and infect with treatment or prevention SARS virus.

Transbilayer helix among the SEQ ID NO:6039 of prediction

Sequence location in the bracket represents nucleus.

Only have when scoring surpasses 500 and just think meaningful.

Interior to external spiral: as to find 43 outside to inside spirals: find 43

Starting from finally, the scoring center starts from the center of finally marking

100(100)118(116)      103   107        94(97)118(112)        291   104

473(473)488(488)      1003  481        400(400)418(415)      243   407

529(532)549(549)      541   539        473(473)488(488)      1113  481

584(584)606(601)      1049  594        523(528)548(548)      285   538

773(773)791(789)      514   782        583(583)606(601)      662   593

1071(1071)1089(1086)  243   1078       776(776)791(791)      1435  783

1121(1121)1137(1137)  459   1130       1068(1071)1089(1086)  370   1078

1679(1679)1696(1696)  404   1686       1121(1121)1137(1137)  455   1130

2098(2102)2119(2116)  509   2109       1679(1679)1696(1694)  340   1686

2145(2145)2160(2160)  797   2153       2098(2098)2119(2116)  678   2109

2206(2209)2224(2224)  2686  2216       2148(2148)2163(2163)  434   2155

2316(2316)2332(2332)  2123  2325       2208(2210)2231(2226)  2389  2219

2335(2339)2358(2354)  2101  2346       2309(2309)2332(2326)  1773  2318

2373(2373)2390(2390)  532   2380      2342(2342)2368(2360)  1666  2353

2597(2600)2615(2615)  307   2607      2373(2373)2390(2390)  254   2380

2753(2753)2770(2768)  2242  2760      2753(2755)2770(2770)  2119  2763

2831(2833)2854(2851)  759   2841      2832(2835)2854(2851)  687   2844

2879(2882)2900(2897)  526   2889      2858(2858)2873(2873)  253   2866

2990(2996)3012(3010)  1289  3003      2879(2882)2899(2899)  400   2889

3024(3024)3042(3039)  2281  3032      2990(2990)3005(3005)  875   2998

3054(3058)3075(3072)  2536  3065      3020(3024)3042(3042)  2795  3032

3105(3109)3127(3123)  2010  3116      3059(3059)3075(3075)  2137  3067

3143(3143)3163(3159)  184   3152      3105(3108)3127(3123)  1902  3115

3253(3255)3272(3272)  319   3262      3142(3145)3162(3162)  540   3152

3346(3346)3366(3366)  203   3356      3343(3351)3366(3366)  496   3358

3375(3375)3392(3392)  305   3384      3437(3437)3453(3453)  848   3444

3438(3438)3455(3453)  1021  3445      3489(3491)3508(3505)  302   3498

3559(3567)3584(3581)  1885  3574      3560(3560)3577(3577)  1460  3569

3589(3589)3606(3604)  2018  3596      3591(3591)3606(3606)  2193  3598

3611(3611)3629(3629)  2304  3621      3610(3610)3627(3627)  1484  3620

3659(3659)3674(3674)  1561  3667      3656(3658)3678(3675)  1240  3668

3756(3758)3777(3774)  2352  3767      3681(3684)3701(3699)  590   3691

3890(3890)3904(3904)  485   3897      3710(3713)3738(3728)  1696  3721

3916(3919)3934(3934)  241   3926      3723(3723)3738(3738)  1670  3730

4035(4035)4051(4051)  335   4044      3760(3760)3777(3775)  2367  3767

4217(4217)4232(4232)  272   4224      3881(3884)3902(3900)  249   3892

4239(4239)4257(4254)  402   4247      4099(4099)4114(4114)  389   4106

                                      4234(4234)4254(4249)  325   4241

                                      4338(4341)4360(4360)  505   4348

Therefore, the present invention includes the polypeptide that contains SEQ ID NO:6039 fragment, wherein said fragment comprises the amino acid sequence that contains one or more hydrophobic transmembrane sequences of identifying above. The present invention includes the polypeptide that contains SEQ ID NO:6039 fragment, wherein said fragment comprises the peptide sequence of following one or more SEQ ID NO:6039: 473-488,529-549,584-606,773-791,2098-2119,2145-2160,2206-2224,2316-2332,2335-2358,2373-2390,2753-2770,2831-2854,2879-2900,2990-3012,3024-3042,3054-3075,3105-3127,3438-3455,3559-3584,3589-3606,3611-3629,3659-3674,3756-3777,473-488,583-606,776-791,2098-2119,2208-2231,2309-2332,2342-2368,2753-2770,2832-2854,2990-3005,3020-3042,3059-3075,3105-3127,3142-3162,3437-3453,3560-3577,3591-3606,3610-3627,3656-3678,3710-3738,3723-3738 and 3760-3777. Preferred this fragment comprises the peptide sequence of following one or more SEQ ID NO:6039: 2206-2224,2316-2332,2335-2358,2753-2770,3024-3042,3054-3075,3105-3127,3589-3606,3611-3629,3756-3777,2208-2231,2753-2770,3020-3042,3059-3075 and 3591-3606. Preferred this fragment comprises the peptide sequence of following one or more SEQ ID NO:6039: 2206-2224 and 3020-3042. The present invention also comprises the polynucleotides of each polypeptide fragment that coding is identified above.

The present invention includes the SARS virus of attenuation, contain interpolation, disappearance or replacement in the polynucleotides of the hydrophobic domains that the SARS virus of wherein said attenuation is identified on coding. The present invention also comprises the method for making the attenuation SARS virus, and the method comprises by the SARS virus genome being added, deletes or replacing the coding that changes one or more hydrophobic domains among the SEQ ID N0:6039 that identifies above undergos mutation SARS virus.

The present invention includes the antibody of one or more hydrophobic domains among the SEQ ID NO:6039 that specific recognition identifies above. The present invention includes can be in conjunction with one or more hydrophobic domains among the SEQ ID NO:6039 that identifies above, disturb its hydrophobicity or make the little molecule of its destruction.

Evaluation during the N-glycosylation site of the SEQ ID NO:6039 of prediction sees the following form.

The position possibility is agreed Nglyc result

  48 NGTC SEQ ID NO：7180  0.6371    (7/9)  +

 389 NHSN SEQ ID NO：7181  0.6132    (6/9)  +

 916 NFSS SEQ ID NO：7182  0.5807    (7/9)  +

1628 NHTK SEQ ID NO：7183  0.5610    (7/9)  +

1696 NKTV SEQ ID NO：7184  0.5297    (5/9)  +

2031 NPTI SEQ ID NO：9764  0.5299    (5/9)  +  WARNING：PRO-X1.

2249 NSSN SEQ ID NO：7185  0.6329    (9/9)  ++

2459 NPTD SEQ ID NO：9765  0.5599    (6/9)  +  WARNING：PRO-X1.

2685 NVSL SEQ ID NO：7186  0.6071    (8/9)  +

4233 NATE SEQ ID NO：7187  0.6144    (7/9)  +

Therefore, the present invention includes the fragment of SEQ ID NO:6039, the amino acid sequence that wherein said fragment comprises contains one or more N-glycosylation sites of identifying above. Preferred this fragment comprises one or more sequences that are selected from lower group: SEQ ID NOS:7180-7187 and 9764-9765. Preferred this fragment comprises amino acid sequence NSSN (SEQ ID NO:7185).

The present invention includes the polypeptide that contains SEQ ID NO:6039 fragment, wherein said fragment does not contain one or more glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this peptide species of encoding.

In table 13, identified the T-epi-position of SEQ ID NO:6039. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:7400-7639; (b) has the amino acid sequence of sequence homogeny with (a) described amino acid sequence. The present invention also comprises coding (a) or (b) polynucleotide sequence of polypeptide. The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:7400-7639, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably.

The invention provides described polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides in mammal (especially people) method that causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, and wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The ORF1a of coronavirus and ORF1b sequence are translated into an ORF1ab polyprotein usually. Ribosomal slip produces an a-1 frameshit in the translation process. A zone of this slip is as follows:

     gggttttacacttagaaacacagtctgtaccgtctgcggaatgtggaaaggttatggctgtagttgtga

+1     G  F  T  L  R  N  T  V  C  T  V  C  G  M  W  K  G  Y  G  C  S  C  D

+3    G  F  Y  T  -  K  H  S  L  Y  R  L  R  N  V  E  R  L  W  L  -  L  -

    ccaactccgcgaacccttgatgcagtctgcggatgcatcaacgtttttaaacgggtttgcggtgtaagt

+1    Q  L  R  E  P  L  M  Q  S  A  D  A  S  T  F  L  N  G  F  A  V  -  V

+3   P  T  P  R  T  L  D  A  V  C  G  C  I  N  V  F  K  R  V  C  G  V  S

    gcagcccgtcttacaccgtgcggcacaggcactagtactg(SEQ ID NO：7224)

+1    Q P V L H R A A Q A L V L(SEQ ID NOS：7225-7226)

+3   A A R L T P C G T G T S T(SEQ ID NOS：7227-7229)

It will produce following translation translation (SEQ ID NOS:7230-7231):

ccaactccgcgaacccttgatgcagtctgcggatgcatcaacgtttttaaacgggtttgcggtgtaagt

 Q  L  R  E  P  L  M  Q  S  A  D  A  S  T  F  L  N R  V  C  G  V  S

Therefore, the present invention includes the polypeptide that contains SEQ ID NO:7232. The polypeptide that the present invention includes contains the amino acid sequence that has the sequence homogeny with SEQ D NO:7232. The present invention includes the fragment that contains SEQ ID NO:7232 polypeptide. The present invention includes diagnostic kit, the polypeptide that contains SEQ ID NO:7232 or its fragment is housed in the box. The present invention includes diagnostic kit, the polynucleotide sequence that contains coding SEQ ID NO:7232 or its fragment is housed in the box. The polypeptide that contains SEQ ID NO:7232 or its fragment in the immunogenic composition that the present invention includes. The present invention includes the antibody that to identify the polypeptide that contains SEQ ID NO:7232 or its fragment.

The present invention also comprises and contains amino acid sequence X₁-X ₂-X ₃Polypeptide, wherein, X₁SEQ ID NO:7233, X₂Be 1-10 amino acid, X₃SEQ ID NO:7234. X₂Can comprise 1-10 amino acid whose any sequence (SEQ ID NOS:7235-7244), but in preferred embodiments, X₂Be selected from: F, FL, FLN, FLNR (SEQ ID NO:7245), FLNRV (SEQ ID NO:7246) and FLNRVC (SEQ ID NO:7247). Preferred X₂SEQ ID NO:7247. These preferred embodiments are shown in SEQ ID NOS:7248-7253.

The polypeptide that the present invention includes contains and described amino acid sequence X₁-X ₂-X ₃Amino acid sequence with sequence homogeny. The present invention includes and contain described amino acid sequence X₁-X ₂-X ₃The fragment of polypeptide. The present invention includes diagnostic kit, be equipped with in the box and contain described amino acid sequence X₁-X ₂-X ₃Or the polypeptide of its fragment. The present invention includes diagnostic kit, encode such amino acid sequences X is housed in the box₁-X ₂-X ₃Or the polynucleotide sequence of its fragment. Contain described amino acid sequence X in the immunogenic composition that the present invention includes₁-X ₂-X ₃Or the polypeptide of its fragment. The present invention includes to identify and contain described amino acid sequence X₁-X ₂-X ₃Or the antibody of the polypeptide of its fragment.

Amino acid sequence X₁-X ₂-X ₃(being SEQ ID NOS:7235-7244) is proved homology on the polyprotein function with murine hepatitis virus. This polyprotein can produce multiple proteins through cutting. Can be by X₁-X ₂-X ₃Polyprotein, wherein X₂Be 6 amino acid (SEQ ID NO:7240), the protein of generation is as follows.

The mouse virus protein	Coordinate in little Murivirus	Coordinate in SEQ ID NO:7240
			Nsp2	3334-3636	3241-3546
Nsp3	3637-3923	3547-3836
			Nsp4	3924-4015 (or 4012)	3837-3919
Nsp5	4016 (or 4013)-4209	3920-4117
			Nsp6	4210-4319	4118-4230
Nsp7	4320-4456	4231-4369
			Nsp9	4457-5384	4370-5301
Nsp10	5385-5984	5302-5902
			Nsp11	5985-6505	5903-6429
Nsp12	6506-6879	6430-6775
			Nsp13	6880-7178	6776-7073

The present invention includes amino acid sequence X₁-X ₂-X ₃The fragment of (being SEQ ID NOS:7235-7244), wherein said fragment comprise one of peptide sequence that table identifies. The present invention also comprises amino acid sequence X₁-X ₂-X ₃Fragment, wherein said fragment is included in the peptide sequence that its N-end has a serine and a glutamine is arranged at its C-end. The present invention also comprises amino acid sequence X₁-X ₂-X ₃Fragment, wherein said fragment is included in the peptide sequence that its N-end has an alanine and a glutamine is arranged at its C-end. The present invention also comprises amino acid sequence X₁-X ₂-X ₃Fragment, wherein said fragment is included in the peptide sequence that its N-end has an asparagine and a glutamine is arranged at its C-end. The present invention also comprises amino acid sequence X₁-X ₂-X ₃Fragment, wherein said fragment is included in the peptide sequence that its N-end has a cysteine and a glutamine is arranged at its C-end. Above-mentioned each fragment can be used for fusion.

The present invention includes diagnostic kit, be equipped with in the box and contain the amino acid sequence X that at least one the preceding paragraph is identified₁-X ₂-X ₃The polypeptide of the fragment of (being SEQ ID NOS:7235-7244). The present invention includes diagnostic kit, the amino acid sequence X that at least one the preceding paragraph of coding is identified is housed in the box₁-X ₂-X ₃The polynucleotide sequence of fragment. The present invention includes immunogenic composition, wherein comprise and contain the amino acid sequence X that a preceding paragraph is identified₁-X ₂-X ₃The polypeptide of fragment. The present invention includes to identify and contain the amino acid sequence X that a preceding paragraph is identified₁-X ₂-X ₃The antibody of polypeptide of fragment.

Amino acid sequence X₁-X ₂-X ₃(X wherein₂6 amino acid) in the N-glycosylation site estimated be the asparagine that is positioned on the following amino acid position through evaluation: 48; 389; 556; 916; 1628; 1696; 1899; 2079; 2249; 2252; 2507; 2685; 3303; 3373; 3382; 3720; 4150; 4233; 4240; 5016; 5280; 5403; 5558; 5650; 5905; 6031; 6130; 6474; 6918; 6973. Therefore, the present invention includes the fragment of SEQ ID NO:7239, wherein said fragment has at least 10 amino acid, and wherein said fragment comprises the asparagine on the following amino acid position of one or more SEQ ID NO:7239: 8; 389; 556; 916; 1628; 1696; 1899; 2079; 2249; 2252; 2507; 2685; 3303; 3373; 3382; 3720; 4150; 4233; 4240; 5016; 5280; 5403; 5558; 5650; 5905; 6031; 6130; 6474; 6918; With 6973.

2 sites, zinc land in the SEQ ID NOS:7235-7244 are positioned at amino acid residue 2102-2112 (SEQ ID NO:7254HGIAAINSVPW) through evaluation. The polypeptide of SEQ ID NOS:7235-7244 will be processed into a plurality of peptides by SARS virus. This zinc land falls into the nsp1 zone of polypeptide. SEQ ID NO:7254 is the target spot of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:7254. The present invention includes the polynucleotides of the peptide sequence of coding SEQ ID NO:7254. The present invention includes screening SEQ ID NO:7254 with the method as inhibitor. Present invention resides in recombinant expressed SEQ ID NO:7254 in the host cell. The present invention includes the fragment of SEQ ID NOS:7235-7244, wherein said fragment comprises SEQ ID NO:7254. The present invention includes the polypeptide that contains SEQ ID NO:7254, wherein said polypeptide and zinc atom are compound. The present invention includes the little molecule that can prevent that zinc ion is combined with SEQ ID NO:7254 polypeptide. The present invention includes fusion, wherein said fusion contains SEQ ID NO:7254.

The polyprotein of SARS virus coding will contain at least two protease domains: papain-sample cysteine (cystein) protease (PLP) and chymotrypsin-picornavirus 3C-sample protease (3CLp). (the PLP domain can have more than one copy). The function of these protease is that this polyprotein is cut into a plurality of less protein. 3C-sample protease is also referred to as " major protein enzyme (main protease) " or Mpro, and itself cuts from polyprotein by himself protease (autoprotease) is active. Usually can be referring to " field of virology " (Fields Virology) (second edition of (volumes) such as Fields, B.N.Raven Press, New York, NY) the 35th chapter, and Anand etc., EMBO Journal (2002) 21 (13): 3213-3224. 3CLp is usually corresponding with the Nsp2 zone of identifying above.

SARS virus 3CLp albumen also take SEQ ID NO:6569 (also have SEQ ID NO:9769) as feature, as shown in figure 15.

Figure 16 also shows SARS virus 3CLp and bird infective bronchitis (IBV; SEQ ID NO:6570), MHV (MHV; SEQ ID NO:6571) and bovine coronavirus (BCoV; The result of 3CLp contrast SEQ ID NO:6572). Therefore, the peptide sequence that the present invention includes contains SEQ ID NO:6569 or its fragment or has the peptide sequence of sequence homogeny with it. The present invention also comprises the polynucleotide sequence of coding SEQ ID NO:6569 or its fragment. The present invention includes the polynucleotide sequence that coding and SEQ ID NO:6569 have the peptide sequence of sequence homogeny.

The present invention also comprises the method for screening SARS virus 3CLp protein inhibitor. In one embodiment, the present invention includes the method for screening SEQ ID NO:6569 inhibitor. Present invention resides in the method for recombinant expressed SARS virus 3CLp albumen in the host cell. The present invention includes the active fragment of the peptide sequence of the recombinant expressed SEQ of containing ID NO:6569 or its enzymolysis or have the method for the peptide sequence of sequence homogeny with it. The present invention includes the little molecule that suppresses or reduce the proteolytic activity of SARS virus 3CLp albumen. The present invention includes the little molecule that suppresses or reduce the proteolytic activity that contains SEQ ID NO:6569 polypeptide.

In Figure 15 and 16, identified the catalytic residue of SARS virus 3CLp. Specifically, catalytic histidine and catalytic cysteine have been identified. This catalytic site is to suppress or to reduce the micromolecular target spot of 3CLp proteinase activity. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6569, wherein said fragment comprises at least a catalytic site. Preferred described catalytic site is selected from catalytic histidine and the catalytic cysteine in Figure 15 and 16. The present invention includes the polynucleotides of coded polypeptide, wherein said polypeptide comprises the fragment of SEQ ID NO:6569, and wherein said fragment comprises at least one catalytic site. Preferred described catalytic site is selected from catalytic histidine and the catalytic cysteine in Figure 15 and 16.

The present invention comprises that also the screening compounds library is to identify the micromolecular method that can suppress SARS virus 3CLp catalytic site. Preferred this 3CLp comprises SEQ ID NO:6569 or its fragment, or has the sequence of sequence homogeny with it. This catalytic site is preferably selected from the catalytic histidine shown in Figure 15 and 16 and catalytic cysteine.

The present invention includes the little molecule that suppresses SARS virus 3CLp catalytic site. Preferred 3CLp comprises SEQ ID NO:6569, or its fragment, or has the sequence of sequence homogeny with it. This catalytic site is preferably selected from the catalytic histidine shown in Figure 15 and 16 and catalytic cysteine.

Identified the residue of SARS virus 3CLp substrate sites among Figure 15 and 16. Specifically, substrate sites is positioned on phenylalanine, tyrosine and the histidine. This substrate sites is to suppress or to reduce the micromolecular target of 3CLp proteinase activity. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6569, wherein said fragment comprises at least one substrate sites. Preferred described substrate sites is selected from substrate phenylalanine, tyrosine and the histidine in Figure 15 and 16. The present invention includes the polynucleotides of coded polypeptide, wherein said polypeptide comprises the fragment of SEQ ID NO:6569, and described fragment comprises at least one substrate sites. Preferred described substrate sites is selected from substrate phenylalanine, tyrosine and the histidine in Figure 15 and 16.

The present invention comprises that also the screening compounds library is to identify the micromolecular method of sealing SARS virus 3CLp substrate sites. Preferred 3CLp comprises SEQ ID NO:6569 or its fragment, or has the sequence of sequence homogeny with it. This substrate sites is preferably selected from substrate phenylalanine, tyrosine and the histidine shown in Figure 15 and 16.

The present invention includes the little molecule that suppresses SARS virus 3CLp substrate sites. Preferred 3CLp comprises SEQ ID NO:6569, or its fragment, or has the sequence of sequence homogeny with it. This substrate sites is preferably selected from substrate phenylalanine, tyrosine and the histidine shown in Figure 15 and 16.

The present invention also comprises the diagnostic kit of the polynucleotides that contain encoding SARS virus 3CLp or its fragment. The 3CLp of preferred SARS virus comprises SEQ ID NO:6569 or its fragment, or has the peptide sequence of sequence homogeny with it. Preferred this fragment comprises the one or more sites that are selected from catalytic site and substrate sites. Preferred this catalytic site is selected from one or more sites of identifying in Figure 15 and 16. Preferred this substrate sites is selected from one or more sites of identifying in Figure 15 and 16.

The present invention also comprises the diagnostic kit that contains SARS virus 3CLp specific antibody or its fragment. Preferred this antibody is the polypeptide that contains SEQ ID NO:6569, or its fragment, or has the specific antibody of the peptide sequence of sequence homogeny with it. Preferred this antibody is the specific antibody that is selected from one or more SARS virus 3CLp site of catalytic site and substrate sites. Preferred this catalytic site is selected from one or more sites of identifying in Figure 15 and 16. Preferred this substrate sites is selected from one or more sites of identifying in Figure 15 and 16.

The present invention includes the polypeptide of the amino acid sequence that contains sequence shown in Figure 25. Amino acid sequence among the Figure 25 that separates with * is SEQ ID NOS:7188 and 7189. The present invention includes to contain with Figure 25 and translate the polypeptide that thing has the amino acid sequence of sequence homogeny. The present invention includes the fragment that contains Figure 25 sequences polypeptide. The present invention includes diagnostic kit, be equipped with in the box and contain the polypeptide that Figure 25 translates thing or its fragment. The present invention includes diagnostic kit, the polynucleotide sequence that coding Figure 25 translates thing or its fragment is housed in the box. The present invention includes and contain the polypeptide that Figure 25 translates thing or its fragment in the immunogenic composition. The present invention includes the antibody that to identify the polypeptide that contains sequence shown in Figure 25 or its fragment. The sequence of Figure 25 is proved to be homology on the ORF1b function with coronavirus.

SEQ ID NO:7188 is the ORF in Figure 25. The present invention includes the polypeptide that contains SEQ ID NO:7188. The present invention includes polypeptide and contain the amino acid sequence that has the sequence homogeny with SEQ ID NO:7188. The present invention includes the fragment of the polypeptide that contains SEQ ID NO:7188. The present invention includes diagnostic kit, the polypeptide that contains SEQ ID NO:7188 or its fragment is housed in the box. The present invention includes the diagnostic kit of the polynucleotide sequence that contains coding SEQ ID NO:7188 or its fragment. The present invention includes immunogenic composition, wherein comprise the polypeptide that contains SEQ ID NO:7188 or its fragment. The present invention includes the antibody that to identify the polypeptide that contains SEQ ID NO:7188 or its fragment.

SEQ ID NO:7190 is the ORF in the SEQ ID NO:7188. The present invention includes polypeptide, its fragment that contains SEQ ID NO:7190 or the polypeptide that has the sequence homogeny with it. The present invention also comprises coding SEQ ID NO:7190, its fragment or has the polynucleotides of the polypeptide of sequence homogeny with it. An example of the polynucleotides of coding SEQ ID NO:7190 is SEQ ID NO:7191.

SEQ ID NO:7188 also contains the ORF that comprises SEQ ID NO:6042. The present invention includes the polypeptide that contains SEQ ID NO:6042. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:6042. The present invention includes the fragment of the polypeptide that contains SEQ ID NO:6042. The present invention includes diagnostic kit, the polypeptide that contains SEQ ID NO:6042 or its fragment is housed in the box. The present invention includes the diagnostic kit of the polynucleotide sequence that contains coding SEQ ID NO:6042 or its fragment. The present invention includes immunogenic composition, wherein comprise the polypeptide that contains SEQ ID NO:6042 or its fragment. The present invention includes the antibody that to identify the polypeptide that comprises SEQ ID NO:6042 or its fragment. SEQ ID NO:6042 be proved to be with coronavirus spike protein function on homology.

The cross-film district of the SEQ ID NO:6042 that estimates is as follows through identifying.

The transbilayer helix of the SEQ ID NO:6042 that estimates

Sequence location in the bracket represents nucleus.

Only have when scoring surpasses 500 and just be considered to meaningful.

Interior to external spiral: as to find 18 outside to inside spirals: find 13

Starting from finally, the scoring center starts from the center of finally marking

1(1)16(16)            959   9         1(1)17(17)            684   10

233(237)257(252)      905   244       222(222)240(237)      238   229

345(347)364(361)      490   354       244(247)264(264)      613   254

345(354)369(369)      420   362       349(355)369(369)      314   362

497(497)513(513)      239   506       496(496)511(511)      488   503

573(573)588(588)      811   580       573(573)591(591)      712   581

645(648)666(663)      302   656       650(652)666(666)      474   659

690(696)714(711)      428   704       674(679)702(696)      190   686

857(860)882(874)      1508  867       691(696)713(711)      210   704

1031(1031)1046(1046)  446   1039      866(868)886(886)      1172  876

1199(1203)1219(1217)  2667  1210      1198(1201)1215(1215)  3221  1208

Spike protein SEQ IID NO:6042 is the polypeptide that is exposed to the surface. Hydrophobic transmembrane may hinder recombinant expression of proteins. Therefore, the present invention includes the polypeptide that contains SEQ ID NO:6042, wherein, one or more hydrophobic regions that the above identifies are removed. The present invention also comprises the polynucleotides of this peptide species of encoding. Present invention resides in recombinant expressed this protein in the host cell. Be used for increasing primer and the fragment thereof of spike protein gene, the fragment such as coding extracellular soluble foreign lands comprises SEQ ID NOS:9753-9763 (Xiao etc. (2003) Biochem Biophys Res Comm 312:1159-1164).

The further feature of SEQ ID NO:6042 is as follows through identifying.

PSORT-protein positioning site estimation

Version 6.4 (WWW)

SEQ ID NO:6042-1255 residue

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 2

N-end position TMS:496, i=2

MTOP: membrane superficial tissue (Hartmann etc.)

I (middle part): 503 charge differences (C-N): 1.0

McG: detection signal peptide sequence (McGeoch)

UR length: 13

UR peak value: 3.28

CR net charge: 0

Distinguish scoring: 8.66

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5): 5.94

Possible cleavage site: 13

As if contain the N-terminus signal sequence that to cut

The amino acid of the mature form of estimating forms:

From 14 calculating

The ALOM New count: 1** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: 0B

ALOM: find cross-film district (Klein etc.)

Counting: 1 value :-12.26 thresholdings :-2.0

Inner possibility=-12.26 cross-film 1202-1218 (1194-1228)

Peripheral possibility=0.16

The ALOM scoring of modifying: 2.55

As if contain Ia type memebrane protein

The kytoplasm tail is from 1219 to 1255 (37 residues)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

(14) maybe can not cut?

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 14

Can not cut? Ipos is made as: 24

Distinguishing of target mtDNA sequence:

Positive electricity (2.18)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

* * inference step: 2

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 24) from: 1 to: 10 the scoring: 8.0

SKL motif (peroxisome signal peptide):

The position: 964 (1255), counting: 1SRL

SKL marks (peroxisome): 0.1

Peroxisome tendentiousness amino acid forms: 1.37

Not from the terminal AAC that counts of N-, the scoring of modification

The peroxidase body protein? state: unclear

AAC marks (peroxisome): 0.079

Lysosomal protein tendentiousness amino acid forms

Scoring: 0.39 state: unclear

GY motif in the Ia type tail? (lysosome)

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

The Ia type is plasmalemma protein choosing of fine quality

Examine the NPXY motif.

Examine the YXRF motif.

Examine the N-myristoylation.

-----final result-----

Plasma membrane---certainty=0.460 (sure)＜success 〉

Microbody (peroxisome)---certainty=0.171 (sure)＜success 〉

Endoplasmic reticulum (film)---certainty=0.100 (sure)＜success 〉

Endoplasmic reticulum (chamber)---certainty=0.100 (sure)＜success 〉

SEQ ID NO:6042 shows to have the terminal signaling zone of N-, is the terminal cytoplasmic structure of zone, cross-film district and the C-territory that is exposed to the surface successively after it. Therefore, the present invention includes having immunogenicity and being exposed to surperficial fragment of SEQ ID NO:6042. Preferred described fragment does not contain last 50 amino acid whose amino acid sequences of SEQ ID NO:6042C-end. Preferred described fragment comprises last 70 the amino acid whose amino acid sequences that do not contain SEQ ID NO:6042C-end. Preferred described fragment does not contain the translation district (transdomain region) of SEQ ID NO:6042. Preferred described fragment does not contain the terminal cytoplasmic structure of the C-territory of SEQ ID NO:6042. Preferred described fragment does not contain the N-terminus signal sequence. Preferred described fragment does not contain the amino acid/11-10 of SEQ ID NO:6042N-end. Preferred described fragment does not contain the amino acid/11-14 of SEQ ID NO:6042N-end. Two oligopeptides fragments that can cause the SEQ ID NO:6042 of anti--furcella antibody are SEQ ID NOS:7398 and 7399, such as (having extra C-terminal cysteine) as described in Xiao etc., 2003, Biochem Biophys Res Comm 312:1159-1164. Yang etc. (2004) have described the brachymemma C-end of spike protein, and the cytoplasmic region part has been removed in this brachymemma, has perhaps also removed cross-film district (Nature 428:561-564).

The variant of the SEQ ID NO:6042 that the present invention is included is SEQ ID NO:9962. Compare with SEQ ID NO:6042, this sequence has replaced Ala with Ser on 581 of residues, and replaces Leu with Phe on 1152 of residues.

The spike protein of coronavirus can be cut into two independently chain S1 and S2. These two chains still can be combined together to form dimer or tripolymer. Therefore, the present invention includes the polypeptide that contains SEQ ID NO:6042, wherein said polypeptide is cut into S1 and S2 domain. The present invention also comprises the polypeptide that contains SEQ ID NO:6042, wherein, the amino acid/11 of N-end-10, preferred amino acid 1-14 is removed, and wherein SEQ ID NO:6042 is cut into S1 and S2 domain. Preferred described polypeptide is trimeric form.

As if spike protein preferably form a α-helixstructure in the cross-film district of protein in the S2 domain. This α-helixstructure is considered to be combined with at least two other spike proteins form tripolymer. The below has identified spiral or the curled regions of spike protein. The coiled coil of estimating SEQ ID NO:6042 (spike protein) is positioned at amino acid 900-1005 and 1151-1185 (referring to Figure 12).

Therefore, the present invention includes the peptide sequence of the fragment that contains SEQ ID NO:6042, wherein said fragment comprises the curled regions of SEQ ID NO:6042. Described fragment preferably includes and is selected from lower group amino acid sequence: 900-1005 amino acids and the 1151-1185 amino acids of SEQ ID NO:6042. The present invention includes the peptide sequence of the fragment that contains SEQ ID NO:6042, wherein said fragment does not comprise the curled regions of SEQ ID NO:6042. Described fragment preferably includes and is selected from lower group amino acid sequence: 900-1005 amino acids and the 1151-1185 amino acids of SEQ ID NO:6042.

Spike protein is considered to play the part of intermediation in coronavirus and mammalian host cell fusion with in infecting. Similar surface protein identifies at least two structural motifs relevant with this fusion in coronavirus spike protein and other virus by analyzing: seven residue repetitive sequence (HR) and film fusogenic peptides, these two motifs are usually located in the S2 domain.

In the extracellular domain of coronavirus S2 domain, usually find to have at least two 4,3 hydrophobic seven residue repetitive sequence (HR) domains. Seven residue repetitive sequence districts (HR1) are usually located at the place of contiguous fusogenic peptide, and second seven residue repetitive sequence district (HR₂) be usually located near the C-end of S2 domain contiguous cross-film anchor point. The feature of seven residue repetitive sequences is coiled coil structures, and the seven residue repetitive sequences of finding in virus surface proteins (such as the coronavirus spike protein) are considered to form the bunchy helical structure relevant with Virus entry. Referring to Bosch etc., (Figure 1B of this list of references shows SARS and 5 kinds of coronavirus HR1 and BR to J.Virology (2003) 77:8801-8811₂The contrast in zone is expressed as " HCov-SARS ").

Seven residue repetitive sequences contain 7 amino acid whose repetitive structures usually, are expressed as a-b-c-d-e-f-g, and wherein, the hydrophobic side chain of residue a and d forms a nonpolar striped (stripe) usually, and has found electrostatic interaction in residue e and g. Position a is the most normal to be Leu, Ile or Ala, and position d often is Leu or Ala. Residue e and g often are Glu or Gln, and Arg and Lys also mainly come across position g. Charged residue is common in b, c and f position, and these residues can contact with solvent. Known these conventional parameters have exception. For example, find sometimes the Pro residue in these 7 amino acid.

HR1 and the HR of MHV strain have been supposed₂Sequence can be assembled into heat resistance oligomerization α rifle bar sample complex, HR1 and HR in the complex₂Spiral moves towards with antiparallel manner. Equally, in this research, HR is proposed₂It is the potent inhibitor of cell entry cell and cell-Fusion of Cells.

HR1 and HR in the SARS virus genome, have been identified₂Sequence. SARS virus HR1 district roughly comprises the amino acid 879-1005 of SEQ ID NO:6042, or it can form the fragment of at least one alpha-helix corner. Preferred described fragment comprises at least 7 (for example, at least 14,21,28,35,42,49 or 56) amino acid residues. SEQ ID NO:7192 comprises the amino acid 879-1005 of SEQ ID NO:6042.

Preferred HR1 fragment comprises the amino acid residue 879-980 of SEQ ID NO:6042. This preferred fragment is SEQ ID NO:7193.

Another preferred HR1 fragment comprises the amino acid residue 901-1005 of SEQ ID NO:6042. This preferred fragment is SEQ ID NO:7194.

SARS virus HR₂The district roughly comprises the amino acid/11 144-1201 of SEQ ID NO:6042, or it can form the fragment of at least one alpha-helix corner. Preferred described fragment comprises at least 7 (for example, at least 14,21,28,35,42,49 or 56) amino acid residues. SEQ ID NO:7195 comprises amino acid/11 144-1201. Preferred HR₂Fragment comprises the amino acid/11 144-1195 of SEQ ID NO:6042. This preferred fragment is SEQ ID NO:7196.

Film fusogenic peptide sequence in the spike protein also is considered to participate in virus and host Fusion of Cells (and infection). Fusogenic peptide contains 16-26 the amino acid residue conservative in virus family of having an appointment usually. These film fusogenic peptides are relatively hydrophobic, and usually are shown as the distribution of asymmetry hydrophobicity when they form the α spiral. They also are rich in alanine and glycine usually.

In coronavirus, identify at least three hydrophobic film fusogenic peptide zones (PEP1, PEP2 and PEP3). Referring to Luo etc., " two effects of conservative hydrophobic region in cell-Fusion of Cells in the mouse coronavirus spike protein " (Roles in Cell-Cell Fusion of Two Conserved Hydrophobic Regions in the Murine Coronavirus Spike Protein), Virology (1998) 244:483-494. Fig. 1 of this paper has shown the contrast of the film fusogenic peptide sequence of MHV, bovine coronavirus, feline infectious peritonitis virus, TGE and IBV. Also can be referring to Bosch etc., " the coronavirus spike protein is I viroid fusion: the 26S Proteasome Structure and Function that merges the core complex " (The Coronavirus Spike Protein is a Class I Virus Fusion Protein:Structural and Functional Characterization of the Fusion Core Complex) Journal of Virology (2003) 77 (16): 8801-8811.

Also identify PEP1 (SEQ ID NO:7197), PEP2 (SEQ ID NO:7198) and PEP3 (SEQ ID NO:7199) sequence in the SARS spike protein.

Think that coronavirus spike protein (and other similar surperficial virus protein) is combined by having gone through conformation change with target cell membrane at acceptor. Think that the result of this conformation change is that one or more hydrophobic film fusogenic peptides are exposed and insert in the target cell membrane. The free energy that discharges when subsequently spike protein refolding becomes its most stable conformation it is believed that in virus and cell membrane merge and plays a significant role.

Available one or more SARS HR sequence, preferred HR₂Or its fragment suppresses cell entry and merges with the mammalian hosts target cell membrane. The invention provides a kind of method that suppresses virus infections, described method comprises the composition that contains one or more SARS HR polypeptide or its fragment. Preferred described composition contains SARS HR₂Sequence.

In another embodiment, the present invention includes and contain SARS HR1 sequence or its fragment and SARS HR₂The composition of sequence or its fragment. Described HR1 and HR₂Sequence can be chosen the formation oligomer that mutually combines wantonly. Described composition can comprise HR1 and HR₂Intermediate structure territory sequence between the domain. Utilize this intermediate sequence can promote oligomerization or other structural interaction between the HR district.

Available methods known in the art recombinant production is used for HR sequence of the present invention. Can modify SARS HR sequence and be beneficial to bacterial expression. Specifically, can modify the HR sequence is beneficial to this recombinant protein is transported to the bacterial host cell surface. For example, can be at the terminal targeting sequencing that adds bacterial membrane protein of the N of restructuring HR sequence. Also can produce for HR sequence of the present invention (seeing below) by chemical synthesis with the known method in this field.

The applicant has identified the structural similarity between the surface protein NadA (and other similar bacterium adhesion protein) of SARS spike protein and Neisseria meningitidis (Neisseria meningitidis), and this specification will be discussed below in more detail. The method that another kind is beneficial to bacterial expression HR sequence is included in handle (stalk) and/or the anchor sequence of the terminal NadA-of adding of the C-sample albumen of restructuring HR sequence. The recombination sequence preparation that preferably will contain bacterium anchor sequence (will more be described in detail its preparation) afterwards in application in outer membrane vesicles. Lacking the recombination sequence of this bacterium anchor sequence can be secreted and separate from supernatant and obtain.

The peptide sequence that the present invention includes comprises First ray and the second sequence, and wherein said First ray comprises the targeting sequencing of bacterial membrane protein, and described the second sequence comprises the HR sequence of coronavirus. Preferred described First ray comprises the targeting sequencing of bacterial adhesion fibroin. More preferably described bacterium adhesion protein is NadA. Preferred described the second sequence comprises HR1, HR₂Or the two. In one embodiment, described the second sequence comprises HR1, HR₂With the intermediate structure territory sequence that exists in the spike protein of natural generation. For example, described the second sequence can comprise coronavirus spike protein fragment, comprises in this fragment to start from HR1 zone N-end and end at HR₂The amino acid of zone C-end.

The present invention also comprises the peptide sequence that contains the first, second, third and the 4th sequence, and wherein said First ray comprises the targeting sequencing of bacterial membrane protein; Wherein said the second sequence comprises the HR sequence of coronavirus; Wherein said the 3rd sequence comprises the handle zone of bacterium adhesion protein; And wherein said the 4th sequence comprises the anchor zone of bacterium adhesion protein. In one embodiment, the First ray that contains leader peptide sequences is removed. In another embodiment, the 3rd sequence that contains the handle zone is removed. In another embodiment, the 4th sequence that contains the anchor zone is removed.

The known method in available this field for example, can link together by the peptide sequence of glycine joint with above-mentioned construction.

The example that can be used for the construction of this bacterial expression system is shown in Figure 50. The peptide sequence of each construction shown in Figure 50 is given in SEQ ID NOS:7200-7206.

7200 leader NadA (2-29)-HR1 (879-980)-6Xgly-HR₂(1144-1195)-handle+anchor NadA (88-405)

7201 leader NadA (1-29)-HR1 (879-980)-6Xgly-HR₂(1144-1196)-handle NadA (88-351)

7202 leader NadA (1-29)-HR1-HR₂(879-1196)-handle+anchor NadA (88-405)

7203 leader NadA (1-29)-HR1-HR₂(879-1196)-handle NadA (88-351)

7204  HR1-HR ₂(879-1196)-handle NadA (88-351)-6 his

7205 leader NadA (1-29)-HR1-HR₂(879-1196)-anchor NadA (351-405)

7206 leader NadA (1-29)-HR1-HR₂(879-1196)

Give one or more abilities that also may disturb coronavirus and host cell membrane to merge in these film fusion sequences. Therefore, the present invention includes the polypeptide of the separation that contains following amino acid sequence: SEQ ID NO:7197, SEQ ID NO:7198 and SEQ ID NO:7199. The present invention also comprises and contains the polypeptide that separates that has the amino acid sequence of sequence homology with following amino acid sequence: SEQ ID NO:7197, SEQ ID NO:7198 and SEQ ID NO:7199.

Two or more in these SARS film fusogenic peptides can be combined together. The present invention includes the composition that contains two kinds of SARS film fusogenic peptides, wherein said peptide is selected from least two kinds of amino acid that are selected from lower group: SEQ ID NO:7197, SEQ ID NO:7198 and SEQ ID NO:7199, or have the sequence of sequence homogeny with it.

Two or more SARS film fusogenic peptides can link together. Therefore, the present invention includes the polypeptide that contains the first amino acid sequence and the second amino acid sequence, wherein said the first and second amino acid sequences are selected from lower group: SEQ ID NO:7197, SEQ ID NO:7198 and SEQ ID NO:7199, or have the sequence of sequence homogeny with it. Preferred described the first amino acid sequence is different SARS film fusogenic peptides with described the second amino acid sequence, and namely they are not identical.

The present invention also comprises the method that treatment or prevention SARS virus infect, and the method comprises and gives one or more above-mentioned SARS film fusogenic peptide compositions.

As mentioned above, described spike protein can form tripolymer. The present invention also comprises the polypeptide that contains trimerization form SEQ ID NO:6042. The present invention includes the composition that contains at least polypeptide, wherein each polypeptide comprises the curling district of alpha-helix of SARS viral spikes at least. Preferred described spike protein comprises SEQ ID NO:6042 or its fragment.

The present invention also comprise contain SARS virus spike protein or its fragment composition, wherein said protein is relevant with cross-film, and wherein said fragment comprises the alpha-helix zone of SARS virus spike protein. Preferred said composition comprises at least three SARS virus spike proteins or its fragment, and wherein said fragment comprises the alpha-helix zone of SARS virus spike protein.

The present invention also comprises the antibody of specific binding SARS virus spike protein trimerization form. Preferred described spike protein comprises SEQ ID NO:6042 or its fragment. The present invention includes the antibody of specific binding SARS virus spike protein trimerization form, wherein said protein is relevant with cross-film.

The present invention also comprises the antibody of specific binding SARS virus spike protein monomeric form or its fragment. The monomeric form of preferred described antibody specific binding SEQ ID NO:6042 or its fragment.

The present invention also comprises interference or destroys the curling little molecule of SARS virus spike protein tripolymer.

The present invention also comprises the attenuation SARS virus as vaccine, and the virus of wherein said attenuation contains the polynucleotides that can not destroy SARS virus spike protein trimerization conformation and inserts, lacks or replace. The present invention also comprises the attenuation SARS virus as vaccine, and the virus of wherein said attenuation contains can not destroy polynucleotides insertion, disappearance or the replacement that SARS virus spike protein alpha-helix forms.

Can make spike protein by recombination method. In one embodiment, described spike protein is expressed in virus-like particle, thereby this protein attachment is on cell membrane. This being attached with is beneficial to the immunogenicity epi-position of offering spike protein. The alpha-helix part of preferred spike protein is combined with cell membrane. Preferred spike protein forms tripolymer in the cross-film district of combination.

The N-glycosylation site of the SEQ ID NO:6042 of prediction is as follows through identifying:

The position possibility is agreed NGlyc result

  29  NYTQ  SEQ ID NO：7207  0.7751      (9/9)  +++

  65  NVTG  SEQ ID NO：7208  0.8090      (9/9)  +++

 109  NKSQ  SEQ ID NO：7209  0.6081      (7/9)  +

 119  NSTN  SEQ ID NO：7210  0.7039      (9/9)  ++

 158  NCTF  SEQ ID NO：7211  0.5808      (7/9)  +

 227  NITN  SEQ ID NO：7212  0.7518      (9/9)  +++

 269  NGTI  SEQ ID NO：7213  0.6910      (9/9)  ++ 

 318  NITN  SEQ ID NO：7214  0.6414      (9/9)  ++

 330  NATK  SEQ ID NO：7215  0.6063      (8/9)  +

 357  NSTF  SEQ ID NO：7216  0.5746      (8/9)  +

 589  NASS  SEQ ID NO：7217  0.5778      (6/9)  +

 602  NCTD  SEQ ID NO：7218  0.6882      (9/9)  ++

 699  NFSI  SEQ ID NO：7219  0.5357      (7/9)  +

 783  NFSQ  SEQ ID NO：7220  0.6348      (9/9)  ++

1080  NGTS  SEQ ID NO：7221  0.5806      (7/9)  +

1116  NNTV  SEQ ID NO：7222  0.5106      (5/9)  +

1176  NESL  SEQ ID NO：7223  0.6796      (9/9)  ++

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6042, wherein said fragment comprises one or more glycosylation sites (SEQ ID NOS:7207-7223) of above identifying. The present invention also comprises the polynucleotides of one or more above-mentioned fragments of encoding. This glycosylation site can be covalently bound to sugar. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6042, wherein said fragment comprises one or more glycosylation sites of above identifying, and wherein said polypeptide in one or more above-mentioned sites by glycosylation.

The O-glycosylation site of prediction is as follows through identifying:

Residue numbering possibility threshold value suggestion

Thr      698      0.8922      0.7696      T

Thr      706      0.9598      0.7870      T

Thr      922      0.9141      0.7338      T

Ser      36       0.8906      0.7264      S

Ser      703      0.8412      0.7676      S

The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6042, wherein said fragment comprises one or more O-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of one or more above-mentioned fragments of encoding. The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6042, wherein said fragment comprises one or more O-glycosylation sites of above identifying, and wherein said polypeptide is covalently bound to sugar at one or more contained glycosylation sites.

The present invention also comprises the polypeptide of the fragment that contains SEQ ID NO:6042, and wherein said fragment comprises one or more N-glycosylation sites of identifying above, and wherein said fragment comprises one or more O-glycosylation sites of identifying above.

The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6042, wherein said fragment does not contain one or more glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this peptide species of encoding.

The phosphorylation site of the SEQ ID NO:6042 of prediction is Ser-346, Tyr-195 and Tyr-723. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6042, wherein said fragment comprises at least 10 amino acid residues, and wherein said fragment comprises one or more amino acid that are selected from lower group: Ser-346, Tyr-195 and Tyr-723. In one embodiment, the amino acid of one or more Ser-346 of being selected from, Tyr-195 and Tyr-723 is phosphorylated.

Xiao etc. (2003) Biochem Biophys Res Comm 312:1159-1164 has described expression and the functional character of spike glycoprotein.

Identified the T-epi-position of SEQ ID NO:6042 in the table 16. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:8041-8280; (b) has the amino acid sequence of sequence homogeny with (a) described amino acid sequence. The present invention also comprises coding (a) or (b) polynucleotide sequence of described polypeptide. The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the two or more SEQ of being accredited as ID NOS:8041-8280, or the polynucleotides of this peptide species of encoding.

Following antigen purposes is preferred: (1) is as the T-cellular antigens; (2) be used between I class MHC albumen (for example I class HLA) and described antigen fragment, generating complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) cause the antigen that CTL replys. Disease and/or infection that this purposes preferably prevents or treat SARS virus to cause. The invention provides polypeptide and making for the application of immune mammal (especially people) with the medicine of opposing SARS virus infection, wherein, described polypeptide as hereinbefore defined.

The invention provides in mammal (especially people) method that causes immune response, the method comprises and gives described mammal polypeptide as hereinbefore defined that wherein said immune response is cell-mediated immune response, and preferred CTL replys. The preferred protectiveness of described immune response or curative.

The present invention includes the polypeptide that contains SEQ ID NO:6040. The polypeptide that the present invention includes contains the amino acid sequence that has the sequence homogeny with SEQ ID NO:6040. The present invention includes the fragment of the polypeptide that contains SEQ ID NO:6040. The present invention includes the polynucleotides of coding SEQ ID NO:6040 or its fragment. The present invention includes the diagnostic kit of the polypeptide that contains SEQ ID NO:6040 or its fragment. The present invention includes the diagnostic kit of the polynucleotide sequence that contains coding SEQ ID NO:6040 or its fragment. The present invention includes the antibody that to identify the polypeptide that contains SEQ ID NO:6040 or its fragment.

SEQ ID NO:6040 is proved homology on the memebrane protein function with coronavirus. The transbilayer helix of the SEQ ID NO:6040 of prediction is as follows through identifying:

The transbilayer helix of prediction

Sequence location in the bracket represents nucleus.

Only have when scoring surpasses 500 and just be considered to meaningful.

Interior to external spiral: as to find 3

From to the scoring center

27(30)       48(45)            1138            38

137(139)     153(153)          486             146

Spiral outside to inside: find 3

From to the scoring center

28(31)       45(45)             819            38

71(73)       90(90)             210            81

136(142)     156(156)           272            149

Has the amino acid region in the highest predicted transmembrane spiral zone from 48 of SEQ ID NO:6040 the 27th amino acids to the. This cross-film district is difficult to recombinant expressed usually. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6040, wherein said fragment does not comprise the amino acid sequence between the 27-48 position. The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6040, wherein said fragment does not comprise the amino acid sequence between the 28-45 position. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

Prediction SEQ ID NO:6040 is imaginary SARS virus protein. Prediction to SEQ ID NO:6040 protein positioning is as follows. Prediction SEQ ID NO:6040 is positioned at one of following position: mitochondrial matrix gap, microbody (peroxisome), nucleus and mitochondrial inner membrane. Estimate that the interior organelle of SEQ ID NO:6040 and infected cell combines.

Therefore, SEQ ID NO:6040 is the target spot of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:6040 or its fragment. The present invention includes the peptide sequence of coding SEQ ID NO:6040 or the polynucleotides of its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6040. Present invention resides in recombinant expressed SEQ ID NO:6040 in the host cell. The present invention includes the little molecule that can prevent that the interior organelle of SEQ ID NO:6040 polypeptide and infected cell from combining. The present invention includes the fusion that contains SEQ ID NO:6040.

PSORT---protein positioning site estimation

Version 6.4 (WWW)

SEQ ID NO:6040 163 residues

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 0

McG: detection signal peptide sequence (McGeoch)

UR length: 9

UR peak value: 1.75

CR net charge: 1

Distinguish scoring :-2.56

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5): 1.94

Possible cleavage site: 53

As if there is not a N-end signal sequence

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

The ALOM New count: 0** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: 0B

ALOM: find cross-film district (K1ein etc.)

Counting: 0 value: 1.32 thresholdings :-2.0

Peripheral possibility=1.32

The ALOM scoring of modifying :-1.16

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 156

    HRSVTI

Distinguishing of target mtDNA sequence:

Unclear (0.88)

Effect: mitochondrial protein

Effect: mitochondrial protein

Effect: mitochondrial protein

Effect: mitochondrial protein

* * inference step: 2

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 156) from: 27 to: 44 the scoring: 5.0

Mitochondrial matrix? scoring: 0.36

SKL motif (peroxisome signal peptide):

The position: 99 (163), counting: 1 SKL

SKL marks (peroxisome): 0.3

Peroxisome tendentiousness amino acid forms :-4.28

The peroxidase body protein? state: unclear

Lysosomal protein tendentiousness amino acid forms

Scoring: 0.02 state: unclear

Lysosomal modification is marked: 0.152

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Find: position: 132 (5) KRKR

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear is revised scoring: 0.60

Nuclear signal peptide state: unclear (0.30)

Examine the CaaX motif.

Examine the N-myristoylation.

Examine the CaaX motif.

-----final result-----

Mitochondrial matrix gap---certainty=0.480 (sure)＜success 〉

Microbody (peroxisome)---certainty=0.300 (sure)＜success 〉

Nuclear---certainty=0.300 (sure)＜success 〉

Mitochondrial inner membrane---certainty=0.188 (sure)＜success 〉

The N-glycosylation site of the SEQ ID NO:6040 of prediction is as follows through identifying.

The position possibility is agreed NGlyc result

2NKTG(SEQ ID NO：7255)    0.7804      (9/9)  +++

106NLTL(SEQ ID NO：7256)  0.6123      (7/9)  +

Therefore, the present invention includes the fragment of SEQ ID NO:6040, wherein said fragment has 10 amino acid at least, and wherein said fragment comprises one or more asparagines at the amino acid position that is selected from SEQ IID NO:6040 2-106 position. The present invention includes the fragment of SEQ ID NO:6040, wherein said fragment comprises the amino acid sequence of the one or more SEQ of being selected from ID NO:7255 and SEQ ID NO:7256. Preferred described fragment comprises amino acid sequence NKTG (SEQ ID NO:7255).

The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6040, wherein said fragment does not comprise one or more glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this peptide species of encoding.

Table 14 has been identified the T-epi-position of SEQ ID NO:6040. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:7640-7800; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the two or more SEQ of being accredited as ID NOS:7640-7800, or the polynucleotides of this peptide species of encoding.

Following antigen purposes is preferred: (1) is as the T-cellular antigens; (2) be used between I class MHC albumen (for example I class HLA) and described antigen fragment, generating complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) cause the antigen that CTL replys. Disease and/or infection that this purposes preferably prevents or treat SARS virus to cause.

The invention provides polypeptide and making for the application of immune mammal (especially people) with the medicine of opposing SARS virus infection, wherein, described polypeptide as hereinbefore defined.

The invention provides in mammal (especially people) method that causes immune response, the method comprises and gives described mammal polypeptide as hereinbefore defined that wherein said immune response is cell-mediated immune response, and preferred CTL replys. The preferred protectiveness of described immune response or curative.

The present invention includes the polypeptide that contains SEQ ID NO:6041. Homology on the verified part of functions with the ORF1ab polyprotein of SEQ ID NO:6041. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:6040. The present invention includes the polypeptide fragment that contains SEQ ID NO:6041. The present invention includes coding and containing the polynucleotide sequence of polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:6041. The present invention includes the polynucleotides that coding contains the fragment of SEQ ID NO:6041 polypeptide.

The present invention includes the diagnostic kit of the polypeptide that SEQ ID NO:6041 or its fragment are housed. The present invention includes the diagnostic kit of the polynucleotide sequence that contains coding SEQ ID NO:6041 or its fragment. The immunogenic composition that the present invention includes contains SEQ ID NO:6041 polypeptide or its fragment. The present invention includes the antibody that to identify the polypeptide that contains SEQ ID NO:6041 or its fragment.

The polyprotein of coronavirus is relevant with enzymatic activity. Therefore, SEQ ID NO:6041 is the target spot of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:6041 or its fragment. The present invention includes the polynucleotides of coding SEQ ID NO:6041 peptide sequence or its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6041. Present invention resides in recombinant expressed SEQ ID NO:6041 in the host cell. The present invention includes and prevent the altogether little molecule of enzymatic activity of SEQ ID NO:6041 polypeptide performance. The present invention includes the fusion that contains SEQ ID NO:6041.

The below has identified predicted transmembrane district or the hydrophobic region of SEQ ID NO:6041. Although the polyprotein of coronavirus is cut into many less protein by proteolysis, the hydrophobic domains in known this polyprotein can mediate film and combine with replication complex, and can significantly change the structure of host cell membrane. Therefore, the hydrophobic domains of polyprotein is the target spot that gene mutation produces attenuation SARS virus vaccine. The target spot of this hydrophobic domains or SARS virus micromolecular inhibitor. This hydrophobic domains also can be used to produce and can infect with treatment or prevention SARS virus by those regional antibody of specific binding.

The transbilayer helix that SEQ ID NO:6041 is possible

Sequence location in the bracket represents nucleus.

Only have when scoring surpasses 500 and just be considered to meaningful.

Interior to external spiral: as to find 18

Start from the center of finally marking

234(234)254(250)       1046 241

256(256)272(270)       252  263

319(319)334(334)       227  327

503(505)522(519)       405  512

613(615)633(629)       619  622

677(679)703(696)       467  689

849(851)869(865)       229  858

1080(1080)1097(1094)   306  1087

1147(1149)1163(1163)   354  1156

1557(1557)1581(1577)   817  1567

1954(1954)1971(1971)   832  1964

2369(2372)2395(2387)   300  2379

2513(2513)2532(2529)   690  2522

Spiral outside to inside: find 14

Start from the center of finally marking

239(239)254(254)       924    247

239(248)272(263)       468    256

311(314)334(328)       267    321

499(503)522(519)       485    512

617(617)634(631)       425    624

849(853)872(872)       572    864

1147(1147)1162(1162)   765    1155

1564(1564)1581(1579)   883    1572

1951(1951)1968(1966)   657    1958

2513(2522)2539(2537)   711    2529

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6041, wherein said fragment comprises the amino acid sequence that contains one or more hydrophobic transmembrane sequences of identifying above. The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6041, wherein said fragment comprises following one or more peptide sequences of SEQ ID NO:6041: 234-254,613-633,1557-1581,1954-1971,2513-2532,239-254,1564-1581,1951-1968,2513-2539. Preferred this fragment comprises following one or more peptide sequences of SEQ ID NO:6041: 234-254 and 239-254. The present invention also comprises the polynucleotides of each polypeptide fragment that coding is identified above.

The present invention includes the SARS virus of attenuation, in the polynucleotides of the hydrophobic domains that the SARS virus of wherein said attenuation is identified interpolation, disappearance or replacement are arranged on coding. The present invention also comprises the method for making the attenuation SARS virus, the method comprises by the SARS virus genome is added, deletes or replaces undergos mutation SARS virus, thereby changes the coding of one or more hydrophobic domains among the SEQ ID NO:6041 that identifies above.

The present invention includes the antibody of one or more hydrophobic domains among the SEQ ID NO:6041 that specific recognition identifies above. The present invention includes in conjunction with one or more hydrophobic domains among the SEQ ID NO:6041 that identifies above, disturb its hydrophobicity or make the little molecule of its destruction.

The N-glycosylation site of the SEQ ID NO:6041 of prediction is identified in following table.

The position possibility is agreed NGlyc result

571NLSH(SEQ ID NO：7257)   0.6598      (8/9)  +

835NTSR(SEQ ID NO：7258)   0.5762      (7/9)  +

958NVTD(SEQ ID NO：7259)   0.7494      (9/9)  ++

1113NISD(SEQ ID NO：7260)  0.7259      (8/9)  +

1205NSTL(SEQ ID NO：7261)  0.6296      (9/9)  ++

1460NVTG(SEQ ID NO：7262)  0.6844      (9/9)  ++

1685NHSV(SEQ ID NO：7263)  0.5181      (5/9)  +

2029NKTT(SEQ ID NO：7264)  0.5423      (5/9)  +

Therefore, the present invention includes the polypeptide of the amino acid sequence fragment that contains SEQ ID NO:6041, wherein said fragment contains one or more N-glycosylation sites of identifying above. The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6041, wherein said fragment comprises one or more sequences among the SEQ ID NOS:7257-7264. Preferred described fragment comprises following one or more sequence: SEQ ID NOS:7257,7259,7260,7261 and 7262. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6041, wherein said fragment does not contain one or more glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this peptide species of encoding.

In table 15, identified the T-epi-position of SEQ ID NO:6041. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:7801-8040; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide that contains the one or more T-epitope sequences that are accredited as SEQ ID NOS:7801-8040, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably.

The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes peptide sequence SEQ ID NO:6043 or its fragment. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:6043. The present invention includes the amino acid sequence of coding SEQ ID NO:6043 or the polynucleotides of its fragment.

The cross-film district of the SEQ ID NO:6043 of prediction is as follows.

Interior to external spiral: as to find 4

From to the scoring center

41(41)            56(56)             1789          49

76(79)            99(99)             2142          89

105(105)          125(125)           1250          115

Spiral outside to inside: find 3

From to the scoring center

41(41)            59(56)              2053         49

76(82)            98(96)              1580         89

103(105)          125(123)            1257         115

Contain the amino acid region of the highest predicted transmembrane helical region from 99 of SEQ ID NO:6043 the 27th amino acids to the. This cross-film district is difficult to recombinant expressed usually. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6043, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. Preferred this fragment does not comprise the amino acid between the 27-48 position. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

Prediction SEQ ID NO:6043 is imaginary SARS virus protein. Prediction to SEQ ID NO:6043 protein positioning is as follows. Prediction SEQ ID NO:6043 is positioned at one of following position: mitochondrial inner membrane, cytoplasmic membrane, golgiosome and mitochondrial inner membrane space. Estimate that the interior organelle of SEQ ID NO:6043 and infected cell combines.

Therefore, SEQ ID NO:6043 is the target spot of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:6043 or its fragment. The present invention includes the peptide sequence of coding SEQ ID NO:6043 or the polynucleotides of its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6043. Present invention resides in recombinant expressed SEQ ID NO:6043 in the host cell. The present invention includes the little molecule that the organelle of polypeptide in infected cell that prevent SEQ ID NO:6043 is combined. The present invention includes the fusion that contains SEQ ID NO:6043.

PSORT---protein positioning site estimation, SEQ ID NO:6043

Version 6.4 (WWW)

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 3

N-end position TMS:40, i=2

MTOP: membrane superficial tissue (Hartmann etc.)

I (middle part): 47 charge differences (C-N): 3.5

McG: detection signal peptide sequence (McGeoch)

UR length: 12

UR peak value: 1.41

CR net charge: 0

Distinguish scoring :-4.67

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5): 3.44

Possible cleavage site: 15

As if there is not a N-end signal sequence

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

The ALOM New count: 2** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: 0B

ALOM: find cross-film district (Klein etc.)

Counting: 2 values :-6.90 thresholdings :-2.0

Inner possibility=-6.90 cross-film 83-99 (78-101)

Inner possibility=-5.04 cross-film 40-56 (37-60)

Peripheral possibility=-0.32

The ALOM scoring of modifying: 1.48

May be IIIb type memebrane protein (Nexo Ccyt)

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 128

    MRCWLC

Distinguishing of target mtDNA sequence:

Unclear (0.76)

Effect: mitochondrial protein

Effect: mitochondrial protein

Effect: mitochondrial protein

Effect: mitochondrial protein

* * inference step: 2

IIIa or IIIb type are that reticulon is preferred

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 128) from: 39 to: 56 the scoring: 11.5

As if contain signal peptide in the mitochondria

Mitochondrial inner membrane? scoring: 0.59

The mitochondrial inner membrane space? scoring: 0.22

SKL motif (peroxisome signal peptide):

The position: 92 (274), counting: 1SHL

SKL marks (peroxisome): 0.3

Peroxisome tendentiousness amino acid forms: 4.78

The peroxidase body protein? state: the positive

Lysosomal protein tendentiousness amino acid forms

Scoring: 1.16 states: unclear

Type iii protein matter may be positioned golgiosome

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

Examine the TMS number (plasma membrane) of III type

Examine the N-myristoylation.

-----final result-----

Mitochondrial inner membrane---certainty=0.664 (sure)＜success 〉

Plasma membrane---certainty=0.600 (sure)＜success 〉

Golgiosome---certainty=0.400 (sure)＜success 〉

Mitochondrial inner membrane space---certainty=0.362 (sure)＜success 〉

N-and the O-glycosylation site of the SEQ ID NO:6043 of prediction are as follows through identifying.

The position possibility is agreed NGlyc result

227NATF(SEQ ID NO：7265)    0.6328    (7/9)    +

Residue numbering possibility threshold value suggestion

Thr    28      0.9095      0.6280T

Thr    32      0.8740      0.6595      T

Thr    34      0.9058      0.6655      T

Thr    170     0.6816      0.6600      T

Thr    267     0.9240      0.5779      T

Thr    268     0.7313      0.5708      T

Thr    269     0.9859      0.5583      T

Thr    270     0.8023      0.5492      T

Ser    27      0.6930      0.6091      S

Ser    252     0.6457      0.5977      S

Therefore, the present invention includes the polypeptide of the amino acid sequence fragment that contains SEQ ID NO:6043, wherein said fragment comprises N-glycosylation site or the O-glycosylation site of identifying above. The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6043, wherein said fragment comprises one or more N-glycosylation sites of identifying above or O-glycosylation site. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6043, wherein said fragment does not contain one or more glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this peptide species of encoding.

In table 17, identified the T-epi-position of SEQ ID NO:6043. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:8281-8486; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:8281-8486, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the polypeptide that contains SEQ ID NO:6044. The present invention includes the fragment that contains SEQ ID NO:6044 or contain the polypeptide that has the sequence of sequence homogeny with SEQ ID NO:206. The present invention includes the polynucleotides of coding SEQ ID NO:6044.

SEQ ID NO:6044 is accredited as imaginary protein. The below has identified hydrophobic region or the cross-film district of the SEQ ID NO:6044 of prediction:

Interior to external spiral: as to find 3

From to the scoring center

1(1)              17(15)              891            8

47(47)            66(63)              221            56

Spiral outside to inside: find 4

From to the scoring center

1(4)              21(19)              599            11

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6044, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. Preferred this fragment does not comprise the amino acid between the 1-19 position. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

Prediction SEQ ID NO:6044 is imaginary SARS virus protein. Prediction to SEQ ID NO:6044 protein positioning is as follows. Prediction SEQ ID NO:6044 is positioned at one of following position: nucleus, mitochondrial matrix, lysosome (chamber) and microbody (peroxisome). Estimate that the interior organelle of SEQ ID NO:6044 and infected cell combines.

Therefore, SEQ ID NO:6044 is the target of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:6044 or its fragment. The present invention includes the peptide sequence of coding SEQ ID NO:6044 or the polynucleotides of its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6044. Present invention resides in recombinant expressed SEQ ID NO:6044 in the host cell. The present invention includes the polypeptide little molecule relevant with the organelle in the infected cell that prevents SEQ ID NO:6044. The present invention includes the fusion that contains SEQ ID NO:6044.

PSORT---protein positioning site estimation, SEQ ID NO:6044

Version 6.4 (WWW)

154 residues

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 0

McG: detection signal peptide sequence (McGeoch)

UR length: 7

UR peak value: 1.06

CR net charge: 1

Distinguish scoring :-7.97

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5) :-3.28

Possible cleavage site: 34

As if there is not a N-end signal sequence

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

The ALOM New count: 0** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: OB

ALOM: find cross-film district (Klein etc.)

Counting: 0 value: 1.43 thresholdings :-2.0

Peripheral possibility=1.43

The ALOM scoring of modifying :-1.19

Gavel: the border of detecting the target mtDNA sequence

Motif seat: 151

    FRKKQV

Distinguishing of target mtDNA sequence:

Unclear (0.46)

* * inference step: 2

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 151) from: 46 to: 50 the scoring: 5.0

Mitochondrial matrix? scoring: 0.36

SKL motif (peroxisome signal peptide):

The position :-1 (154), counting: 0

Peroxisome tendentiousness amino acid forms: 0.61

The peroxidase body protein? state: unclear

AAC marks (peroxisome): 0.149

Lysosomal protein tendentiousness amino acid forms

Scoring: 0.81 state: unclear

Lysosomal modification is marked: 0.231

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Find: position: 134 (3) KHKK

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Find: position: 136 (3) KK VSTNLCTHSF RKKQV

Final Robbins scoring (nuclear): 0.60

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear is revised scoring: 0.90

Nuclear signal state: positive electricity (0.70)

Examine the CaaX motif.

Examine the N-myristoylation.

Examine the CaaX motif.

-----final result-----

Nuclear---certainty=0.880 (sure)＜success 〉

Mitochondrial matrix gap---certainty=0.360 (sure)＜success 〉

Lysosome (chamber)---certainty=0.231 (sure)＜success 〉

Microbody (peroxisome)---certainty=0.149 (sure)＜success 〉

Identify the O-glycosylation site of the prediction of SEQ ID NO:6044 at No. 4 residues.

Residue numbering possibility threshold value suggestion

Thr     4         0.6839        0.6484      T

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6044 amino acid sequence, wherein said fragment comprises the O-glycosylation site of identifying above. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6044, wherein said fragment does not contain one or more glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this peptide species of encoding.

In table 18, identified the T-epi-position of SEQ ID NO:6044. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:8487-8665; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises one or more being accredited as that contains SEQ ID NOS:8487-8665

The polypeptide of T-epitope sequences, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the peptide sequence that contains SEQ ID NO:6045. The present invention includes and contain the peptide sequence that has the amino acid sequence of sequence homogeny with SEQ ID NO:6045. The present invention includes the peptide sequence of the fragment that contains SEQ ID NO:6045. The present invention includes arbitrary polynucleotide sequence in these polypeptide of coding.

SEQ ID NO:6045 be proved with the envelope protein of coronavirus or membranelle protein function on homology. The present invention includes diagnostic kit, the polypeptide that contains SEQ ID NO:6045 or its fragment is housed in the box. The present invention includes diagnostic kit, the polynucleotides of coding SEQ ID NO:6045 or its fragment are housed in the box. The present invention includes the immunogenic composition that contains SEQ ID NO:6045 or its fragment. The present invention includes the antibody that specific recognition contains the polypeptide of SEQ ID NO:6045 or its fragment.

The below has identified the cross-film district of the SEQ ID NO:6045 of prediction:

Interior to external spiral: as to find 1

From to the scoring center

17(19)             33(33)         2881          26

Spiral outside to inside: find 1

From to the scoring center

17(17)             34(34)         2981          27

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6045, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. Preferred this fragment does not comprise the amino acid between the 17-34 position. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above. In one embodiment, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6045, wherein said fragment does not comprise the amino acid residue of SEQ ID NO:6045 1-34 position.

The protein positioning site of the SEQ ID NO:6045 of prediction is as follows.

PSORT---protein positioning site estimation, SEQ ID NO:6045

Version 6.4 (WWW)

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 2

N-end position TMS:17, i=1

MTOP: membrane superficial tissue (Hartmann etc.)

I (middle part): 24 charge differences (C-N): 2.0

McG: detection signal peptide sequence (McGeoch)

UR length: 29

UR peak value: 3.40

CR net charge :-2

Distinguish scoring: 13.07

GvH: detection signal peptide sequence (yon Heijne)

Signal peptide scoring (3.5): 4.37

Possible cleavage site: 32

The positive value of mtop

As if the N-terminus signal sequence that can not cut arranged

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

The ALOM New count: 1** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: OB

ALOM: find cross-film district (Klein etc.)

Counting: 1 value :-15.12 thresholdings :-2.0

Inner possibility=-15.12 cross-film 17-33 (8-44)

Peripheral possibility=0.47

The ALOM scoring of modifying: 3.12

Ib type (Nexo Ccyt) memebrane protein seemingly

The kytoplasm tail is from 34 to 76 (44 residues)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

(6) maybe can not cut?

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 6

Can not cut? Ipos is made as: 16

Distinguishing of target mtDNA sequence:

Unclear (0.19)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

* * inference step: 2

The relative position of tail end: 44%

Memebrane protein has the signal peptide that can not cut usually at ER

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 16) from: 70 to: 99 the scoring: 21.5

As if contain signal peptide in the mitochondria

SKL motif (peroxisome signal peptide):

The position :-1 (76), counting: 0

Peroxisome tendentiousness amino acid forms :-4.11

The peroxidase body protein? state: feminine gender

Lysosomal protein tendentiousness amino acid forms

Scoring: 0.68 state: unclear

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

Examine Ib type kytoplasm tail (plasma membrane)

Examine the NPXY motif.

Examine the YXRF motif.

Examine the N-myristoylation.

-----final result------

Plasma membrane---certainty=0.730 (sure)＜success 〉

Endoplasmic reticulum (film)---certainty=0.640 (sure)＜success 〉

Endoplasmic reticulum (chamber)---certainty=0.100 (sure)＜success 〉

The outside---certainty=0.100 (sure)＜success 〉

Identify the N-glycosylation site of prediction at 48 and No. 66 residues of SEQ ID NO:6045.

The position possibility is agreed NGlyc result

48NVSL    0.6514    (9/9)    ++        (SEQ ID NO：7266) 

66NSSE    0.5880    (7/9)    +         (SEQ ID NO：7267)

Therefore, the present invention includes the polypeptide of the amino acid sequence fragment that contains SEQ ID NO:6045, wherein said fragment comprises one or more N-glycosylation sites of identifying above. The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6045, wherein said fragment does not contain one or more N-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

The present invention includes the polypeptide of the fragment that contains SEQ ID NO:6045, wherein said fragment does not comprise one or more glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this peptide species of encoding.

In table 19, identified the T-epi-position of SEQ ID NO:6045. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:8666-8820; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:8666-8820, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the peptide sequence that contains SEQ ID NO:6046. The present invention includes and contain the peptide sequence that has the amino acid sequence of sequence homogeny with SEQ ID NO:6046. The present invention includes the peptide sequence of the fragment that contains SEQ ID NO:6046. The present invention includes arbitrary polynucleotides in these polypeptide of coding.

Homology on the stromatin function of SEQ ID NO:6046 and coronavirus. The present invention includes diagnostic kit, the polypeptide that contains SEQ ID NO:6046 or its fragment is housed in the box. The present invention includes diagnostic kit, the polynucleotides of coding SEQ ID NO:6046 or its fragment are housed in the box. The present invention includes the immunogenic composition that contains SEQ ID NO:6046 or its fragment. The present invention includes the antibody that specific recognition contains the polypeptide of SEQ ID NO:6046 or its fragment.

The cross-film district that has identified the SEQ ID NO:6046 that predicts is as follows:

Interior to external spiral: as to find 3

From to the scoring center

21(21)        38(36)        2412        29

51(53)        69(69)        2645        60

74(82)        96(96)        2464        89

Spiral outside to inside: find 3

From to the scoring center

18(21)        38(38)        2363        28

52(52)        67(67)        2363        60

76(76)        95(92)        2605        84

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6046, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. Preferred this fragment does not comprise the amino acid that is selected from between the upper/lower positions: 18-38,52-67 and 76-95. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

The protein positioning of the SEQ ID NO:6046 of prediction is as follows.

PSORT---protein positioning site estimation

Version 6.4 (WWW)

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 3

N-end position TMS:21, i=1

MTOP: membrane superficial tissue (Hartmann etc.)

I (middle part): 28 charge differences (C-N): 6.0

McG: detection signal peptide sequence (McGeoch)

UR length: 1

UR peak value: 3.16

CR net charge :-3

Distinguish scoring: 2.21

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5): 4.29

Possible cleavage site: 39

The positive value of mtop

As if the N-terminus signal sequence that can not cut arranged

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

But in ALOM, detect cleavable signal peptide?: OB

ALOM: find cross-film district (Klein etc.)

Counting: 3 values :-7.64 thresholdings: 0.5

Inner possibility=-7.64 cross-film 21-37 (18-39)

Inner possibility=-7.59 cross-film 50-66 (43-72)

Inner possibility=-5.04 cross-film 79-95 (72-99)

Peripheral possibility=2.38

The ALOM scoring of modifying: 2.13

May be IIIb type memebrane protein (Nexo Ccyt)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

(2) maybe can not cut?

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 2

Can not cut? Ipos is made as: 12

Distinguishing of target mtDNA sequence:

Negative (4.16)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

* * inference step: 2

IIIa or IIIb type are that reticulon is preferred

Memebrane protein has the signal peptide that can not cut usually at ER

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

SKL motif (peroxisome signal peptide):

The position :-1 (221), counting: 0

Peroxisome tendentiousness amino acid forms: 5.01

The peroxidase body protein? state: unclear

Lysosomal protein tendentiousness amino acid forms

Scoring: 2.30 states: the positive

Type iii protein matter may be positioned golgiosome

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

Examine the TMS number (plasma membrane) of III type

Examine the N-myristoylation.

------final result------

Endoplasmic reticulum (film)---certainty=0.685 (sure)＜success 〉

Plasma membrane---certainty=0.640 (sure)＜success 〉

Golgiosome---certainty=0.460 (sure)＜success 〉

Endoplasmic reticulum (chamber)---certainty=0.100 (sure)＜success 〉

Identify the N-glycosylation site of a prediction at No. 4 residues of SEQ ID NO:6046:

The prediction of N-glycosylation site

The position possibility is agreed NGlyc result

4NGTI     0.8430    (9/9)     +++          (SEQ ID NO：7268)

Therefore, the present invention includes the polypeptide of the amino acid sequence fragment that contains SEQ ID NO:6046, wherein said fragment comprises one or more N-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

The present invention also comprises the polypeptide of the fragment that contains amino acid sequence SEQ ID NO:6046, and wherein said fragment does not contain one or more N-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

A variant that is included in the SEQ ID NO:6046 within the scope of the invention is SEQ ID NO:9963. Compare with SEQ ID NO:6046, this sequence Val on 72 residues has replaced Ala.

In table 20, identified the T-epi-position of SEQ ID NO:6046. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:8821-9018; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:8821-9018, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the peptide sequence that contains SEQ ID NO:6047 or its fragment or have the amino acid sequence of sequence homogeny with it. The cross-film district of the SEQ ID NO:6047 of prediction is as follows through identifying.

Interior to external spiral: as to find 2

From to the scoring center

7(10)        29(27)        729        17

21(24)       41(41)        640        34

Spiral outside to inside: find 2

From to the scoring center

4(4)         22(19)        874         2

22(24)       41(41)        499         31

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6047, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. Preferred this fragment does not comprise the amino acid that is selected from between the upper/lower positions: 4-22 and 22-41. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

Prediction SEQ ID NO:6047 is imaginary SARS virus protein. Prediction to SEQ ID NO:6047 protein positioning is as follows. Prediction SEQ ID NO:6047 is positioned at one of following position: cytoplasmic membrane, endoplasmic reticulum, golgiosome and microbody (peroxisome). Organelle in expectation SEQ ID NO:6047 and the infected cell combines or is relevant with the cell entry host cell.

Therefore, SEQ ID NO:6047 is the target of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:6047 or its fragment. The present invention includes the peptide sequence of coding SEQ ID NO:6047 or the polynucleotides of its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6047. Present invention resides in recombinant expressed SEQ ID NO:6047 in the host cell. The present invention includes that the organelle of polypeptide in infected cell that prevents SEQ ID NO:6047 is combined or with the interactional little molecule of host cell membrane. The present invention includes the fusion that contains SEQ ID NO:6047. The protein positioning of the SEQ ID NO:6047 of prediction is as follows.

PSORT---protein positioning site estimation

Version 6.4 (WWW)

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 1

N-end position TMS:2, i=1

MTOP: membrane superficial tissue (Hartmann etc.)

I (middle part): 9 charge differences (C-N): 0.5

McG: detection signal peptide sequence (McGeoch)

UR length: 6

UR peak value: 3.08

CR net charge: 0

Distinguish scoring: 5.12

GvH: detection signal peptide sequence (von Hei jne)

Signal peptide scoring (3.5) :-4.45

Possible cleavage site: 34

As if the N-terminus signal sequence that can not cut arranged

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

The ALOM New count: 1** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: 0B

ALOM: find cross-film district (Klein etc.)

Counting: 1 value :-2.44 thresholdings :-2.0

Inner possibility=-2.44 cross-film 2-18 (1-20)

Peripheral possibility=1.22

The ALOM scoring of modifying: 0.59

II type (Ncyt Cexo) memebrane protein seemingly

The kytoplasm tail is from 1 to 1 (1 residue)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

(5) maybe can not cut?

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 5

Can not cut? Ipos is made as: 15

Distinguishing of target mtDNA sequence:

Unclear (1.48)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

* * inference step: 2

The relative position of kytoplasm tail: 1%

The ER memebrane protein is larger value (＞30%) preferably

Memebrane protein has the signal peptide that can not cut usually at ER

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 15) from: 64 to: 93 the scoring: 30.0

As if contain signal peptide in the mitochondria

SKL motif (peroxisome signal peptide):

The position :-1 (63), counting: 0

Peroxisome tendentiousness amino acid forms: 1.91

The peroxidase body protein? state: unclear

AAC marks (peroxisome): 0.161

Lysosomal protein tendentiousness amino acid forms

Scoring: 0.04 state: unclear

Examine the consensus sequence of golgiosome

Examine the consensus sequence of golgiosome

Examine the kytoplasm tail (golgiosome) of II type

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

Examine the mitochondrial signal peptide (plasma membrane) of II type

The II type is that plasmalemma protein is preferred

Examine the NPXY motif.

Examine the YXRF motif.

Examine the N-myristoylation.

-----final result-----

Plasma membrane---certainty=0.685 (sure)＜success 〉

Endoplasmic reticulum (film)---certainty=0.640 (sure)＜success 〉

Golgiosome---certainty=0.370 (sure)＜success 〉

Microbody (peroxisome)---certainty=0.161 (sure)＜success 〉

In table 21, identified the T-epi-position of SEQ ID NO:6047. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:9019-9131; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:9019-9131, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the polypeptide that contains SEQ ID NO:6048 or its fragment or have the amino acid sequence of sequence homogeny with it. The cross-film district of the SEQ ID NO:6048 of prediction is as follows through identifying.

Interior to external spiral: as to find 2

From to the scoring center

3(3)           18(18)           1857        10

100(100)       117(115)         2904        107

Spiral outside to inside: find 2

From to the scoring center

1(1)            15(15)          1299        8

100(100)        117(115)        3009        107

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6048, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. Preferred this fragment does not comprise and is selected from following locational amino acid: 1-15 and 100-117. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

Prediction SEQ ID NO:6048 is imaginary SARS virus protein. Prediction to SEQ ID NO:6048 protein positioning is as follows. Prediction SEQ ID NO:6048 is positioned at one of following position: cytoplasmic membrane, lysosome (film), microbody (peroxisome) and endoplasmic reticulum (film). SEQ ID NO:6048 may be relevant with the organelle in the infected cell or when the cell entry host cell and the host cell cytoplasmic membrane interact.

Therefore, SEQ ID NO:6048 is the target spot of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:6048 or its fragment. The present invention includes the peptide sequence of coding SEQ ID NO:6048 or the polynucleotides of its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6048. Present invention resides in recombinant expressed SEQ ID NO:6048 in the host cell. The present invention includes that the organelle of polypeptide in infected cell that prevents SEQ ID NO:6048 is combined or with the interactional little molecule of host cell membrane. The present invention includes the fusion that contains SEQ ID NO:6048. The protein positioning of the SEQ ID NO:6048 of prediction is as follows.

PSORT---protein positioning site estimation

Version 6.4 (WWW)

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 2

N-end position TMS:3, i=2

MTOP: membrane superficial tissue (Hartmann etc.)

I (middle part): 10 charge differences (C-N) :-2.5

McG: detection signal peptide sequence (McGeoch)

UR length: 13

UR peak value: 3.38

CR net charge: 1

Distinguish scoring: 10.02

GvH: detection signal peptide sequence (yon Heijne)

Signal peptide scoring (3.5): 2.56

Possible cleavage site: 15

As if contain the N-terminus signal sequence that to cut

The amino acid of the mature form of estimating forms:

Calculate 16 from the position

The ALOM New count: 2** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: 1B

ALOM: find cross-film district (Klein etc.)

Counting: 1 value :-14.75 thresholdings :-2.0

Inner possibility=-14.75 cross-film 101-117 (95-120)

Peripheral possibility=6.63

The ALOM scoring of modifying: 3.05

As if contain Ia type memebrane protein

The kytoplasm tail is from 118 to 122 (5 residues)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

(15) maybe can not cut?

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 15

Can not cut? Ipos is made as: 25

Distinguishing of target mtDNA sequence:

Unclear (0.73)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

* * inference step: 2

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 25) from: 3 to: 12 the scoring: 8.5

SKL motif (peroxisome signal peptide):

The position :-1 (122), counting: 0

Peroxisome tendentiousness amino acid forms: 2.46

Not from the terminal AAC that counts of N-, the scoring of modification

The peroxidase body protein? state: unclear

AAC marks (peroxisome): 0.115

Lysosomal protein tendentiousness amino acid forms

Scoring :-0.40 state: feminine gender

GY motif in the Ia type tail? (lysosome)

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ DingWall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

The Ia type is plasmalemma protein choosing of fine quality

Examine the NPXY motif.

Examine the YXRF motif.

Examine the N-myristoylation.

Examine the GPI anchor.

GPI-anchor (0.85) seemingly

-----final result-----

Plasma membrane---certainty=0.919 (sure)＜success 〉

Lysosome (film)---certainty=0.200 (sure)＜success 〉

Microbody (peroxisome)---certainty=0.115 (sure)＜success 〉

Endoplasmic reticulum (film)---certainty=0.100 (sure)＜success 〉

In table 22, identified the T-epi-position of SEQ ID NO:6048. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:9132-9308; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:9132-9308, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the polypeptide that contains SEQ ID NO:6049 or its fragment or have the amino acid sequence of sequence homogeny with it. Cross-film district or the hydrophobic region of the SEQ ID NO:6049 of prediction are as follows through identifying.

Interior to external spiral: as to find 1

From to the scoring center

13(13)         30(28)          3532            20

Spiral outside to inside: find 1

From to the scoring center

9(11)          29(26)          3395            19

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6049, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

Prediction SEQ ID NO:6049 is imaginary SARS virus protein. Prediction to SEQ ID NO:6049 protein positioning is as follows. Prediction SEQ ID NO:6049 is positioned at one of following position: the outside, microbody (peroxisome), endoplasmic reticulum (film) and endoplasmic reticulum (chamber). The highest location level shows that SEQ ID NO:6049 is positioned at the extracellular. Therefore, SEQ ID NO:6049 may be the protein that is exposed to the surface.

Therefore, SEQ ID NO:6049 can be used to cause the immunogenic composition of the immune response of anti-SARS virus. It also can be used for producing the specific antibody of SARS virus. This antibody can be used for treating or preventing the method for SARS virus infection. This antibody also can be used for diagnostic test and comes whether to exist in the identification of organism sample SARS virus.

The present invention includes the polypeptide that contains SEQ ID NO:6049 or its fragment. The present invention includes the peptide sequence of coding SEQ ID NO:6049 or the polynucleotides of its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6049. Present invention resides in recombinant expressed SEQ ID NO:6049 in the host cell. The present invention includes the fusion that contains SEQ ID NO:6049. The protein positioning of the SEQ ID NO:6049 of prediction is as follows.

PSORT---protein positioning site estimation

Version 6.4 (WWW)

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 1

N-end position TMS:11, i=1

MTOP: membrane superficial tissue (Hartmann etc.)

I (middle part): 18 charge differences (C-N) :-2.0

McG: detection signal peptide sequence (McGeoch)

UR length: 24

UR peak value: 3.69

CR net charge :-2

Distinguish scoring: 13.56

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5): 0.52

Possible cleavage site: 25

As if contain the N-terminus signal sequence that to cut

The amino acid of the mature form of estimating forms:

Calculate 26 from the position

The ALOM New count: 1** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: 1B

ALOM: find cross-film district (Klein etc.)

Counting: 0 value: 14.80 thresholdings :-2.0

Peripheral possibility=14.80

The ALOM scoring of modifying :-3.86

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

(2) maybe can not cut?

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 2

Can not cut? Ipos is made as: 12

Distinguishing of target mtDNA sequence:

Unclear (1.42)

Effect: vesicle path

Effect: vesicle path

Effect: vesicle path

* * inference step: 2

KDEL counting: 0

Potential N-glycosylation site number: 0

Outward: scoring 0.800

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 12) from: 44 to: 73 the scoring: 30.0

As if contain signal peptide in the mitochondria

SKL motif (peroxisome signal peptide):

The position :-1 (44), counting: 0

Peroxisome tendentiousness amino acid forms: 9.47

Not from the terminal AAC that counts of N-, the scoring of modification

The peroxidase body protein? state: unclear

AAC marks (peroxisome): 0.320

Lysosomal protein tendentiousness amino acid forms

Scoring :-6.47 states: feminine gender

NX (S/T) motif number: 0

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

Examine the CaaX motif.

Examine the N-myristoylation.

Examine the CaaX motif.

-----final result-----

The outside---certainty=0.820 (sure)＜success 〉

Microbody (peroxisome)---certainty=0.320 (sure)＜success 〉

Endoplasmic reticulum (film)---certainty=0.100 (sure)＜success 〉

Endoplasmic reticulum (chamber)---certainty=0.100 (sure)＜success 〉

In table 23, identified the T-epi-position of SEQ ID NO:6049. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:9309-9437; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:9309-9437, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the polypeptide that contains SEQ ID NO:6050 or its fragment or have the amino acid sequence of sequence homogeny with it. Cross-film district or the hydrophobic region of the SEQ ID NO:6050 of prediction are as follows through identifying.

Interior to external spiral: as to find 1

From to the scoring center

13            (15)32(30)        558        23

Spiral outside to inside: find 1

From to the scoring center

16(16)         30(30)           364        23

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6050, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

Prediction SEQ ID NO:6050 is imaginary SARS virus protein. Prediction to SEQ ID NO:6050 protein positioning is as follows. Prediction SEQ ID NO:6050 is positioned at one of following position: gap between lysosome (chamber), mitochondrial matrix gap, mitochondrial inner membrane and mitochondrial membrane. SEQ ID NO:6050 may combine with organelle in the infected cell in virus replication cycle period.

Therefore, SEQ ID NO:6050 is the target of screening SARS virus chemical inhibitor. The present invention includes the polypeptide that contains SEQ ID NO:6050 or its fragment. The present invention includes the peptide sequence of coding SEQ ID NO:6050 or the polynucleotides of its fragment. The present invention includes the method for the inhibitor of screening SEQ ID NO:6050. Present invention resides in recombinant expressed SEQ ID NO:6050 in the host cell. The present invention includes prevent that the organelle of SEQ ID NO:6050 polypeptide in infected cell is combined or with the interactional little molecule of host cell membrane. The present invention includes the fusion that contains SEQ ID NO:6050. The protein positioning of the SEQ ID NO:6050 of prediction is as follows.

PSORT---protein positioning site estimation

Version 6.4 (WWW)

MYSEQ 84 residues

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 0

McG: detection signal peptide sequence (McGeoch)

UR length: 3

UR peak value: 1.46

CR net charge: 2

Distinguish scoring :-5.73

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5) :-0.12

Possible cleavage site: 29

As if there is not a N-end signal sequence

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

The ALOM New count: 0** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: 0B

ALOM: find cross-film district (Klein etc.)

Counting: 0 value: 8.43 thresholdings :-2.0

Peripheral possibility=8.43

The ALOM scoring of modifying :-2.59

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 61

    ARCWYL

Distinguishing of target mtDNA sequence:

Positive electricity (1.66)

Effect: mitochondrial protein

Effect: mitochondrial protein

Effect: mitochondrial protein

Effect: mitochondrial protein

* * inference step: 2

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

(Gavel position 61) from: 52 to: 58 the scoring: 6.0

Mitochondrial matrix? scoring: 0.38

SKL motif (peroxisome signal peptide):

The position :-1 (84), counting: 0

Peroxisome tendentiousness amino acid forms: 1.47

The peroxidase body protein? state: unclear

AAC marks (peroxisome): 0.263

Lysosomal protein tendentiousness amino acid forms

Scoring: 2.86 states: the positive

Lysosomal modification is marked: 0.850

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear signal state: negative (0.00)

Examine the CaaX motif.

Examine the N-myristoylation.

Examine the CaaX motif.

-----final result-----

Lysosome (chamber)---certainty=0.850 (sure)＜success 〉

Mitochondrial matrix gap---certainty=0.544 (sure)＜success 〉

Mitochondrial inner membrane---certainty=0.266 (sure)＜success 〉

Mitochondrial inner membrane space---certainty=0.266 (sure)＜success 〉

Identify the N-glycosylation site of the prediction of SEQ ID NO:6050 at No. 43 residues:

The position possibility is agreed NGlyc result

43NVTI    0.6713       (9/9)     ++        (SEQ ID NO：7269)

Therefore, the present invention includes the polypeptide of the amino acid sequence fragment that contains SEQ ID NO:6050, wherein said fragment comprises one or more N-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

The present invention also comprises the polypeptide of the fragment that contains amino acid sequence SEQ ID NO:6050, and wherein said fragment does not contain one or more N-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this fragment of encoding.

In table 24, identified the T-epi-position of SEQ ID NO:6050. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:9438-9538; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:9438-9538, or the polynucleotides of this peptide species of encoding.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the peptide sequence that contains SEQ ID NO:6051 or its fragment or have the amino acid sequence of sequence homogeny with it. The present invention includes the peptide sequence that contains SEQ ID NO:6052 or its fragment or have the amino acid sequence of sequence homogeny with it.

SEQ ID NO:6051 and SEQ ID NO:6052 are proved homology on the nucleocapsid protein function with coronavirus. The present invention includes diagnostic kit, the polypeptide that contains SEQ ID NO:6051, SEQ ID NO:6052 or its fragment is housed in the box. The present invention includes diagnostic kit, the polynucleotides of coding SEQ ID NO:6051, SEQ ID NO:6052 or its fragment are housed in the box. The present invention includes the immunogenic composition that contains SEQ ID NO:6051, SEQ ID NO:6052 or its fragment. The present invention includes the antibody that to identify the polypeptide that comprises SEQ ID NO:6051, SEQ ID NO:6052 or its fragment.

Prediction SEQ ID NO:6051 is at Ser-79; Thr-92; Ser-106; Thr-116; Thr-142; Ser-184; Ser-188; Ser-202; Ser-236; Thr-248; Ser-251; Ser-256; Be phosphorylated on the Thr-377. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6051, wherein said fragment comprises following one or more amino acid residues of SEQ ID NO:6051: Ser-79; Thr-92; Ser-106; Thr-116; Thr-142; Ser-184; Ser-188; Ser-202; Ser-236; Thr-248; Ser-251; Ser-256; Thr-377. The present invention also comprises the polypeptide of the fragment that contains SEQ ID NO:6051, and wherein said fragment does not comprise following one or more amino acid residues of SEQ ID NO:6051: Ser-79; Thr-92; Ser-106; Thr-116; Thr-142; Ser-184; Ser-188; Ser-202; Ser-236; Thr-248; Ser-251; Ser-256; Thr-377. Other two relevant fragments of N albumen (for example being used for immunoprecipitation) are SEQ ID NOS:9783﹠9784, and they are rich in lysine and can be used for distinguishing SARS virus and other coronavirus.

The cross-film district of the SEQ ID NO:6051 of prediction is as follows through identifying.

Interior to external spiral: as to find 1

From to the scoring center

304(304)        323(319)     495            312

Spiral outside to inside: find 1

From to the scoring center

304(304)         319(319)    597            312

Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:6051, wherein said fragment does not comprise one or more hydrophobic amino acid sequences of identifying above. The present invention also comprises the polynucleotide sequence of any polypeptide that coding is identified above.

The protein positioning of the SEQ ID NO:6051 of prediction is as follows. Estimate that SEQ ID NO:6051 is positioned near nucleus, lysosome (chamber), mitochondrial matrix gap and the microbody (peroxisome). The highest location level is positioned near the nucleus. The nucleocapsid protein of known coronavirus is in conjunction with viral RNA. Think that also the coronavirus nucleocapsid protein is very important for cell-mediated immunity. Therefore, the present invention includes the polynucleotides that contain SEQ ID NO:6051. The present invention also comprises viral vectors or the particle that is fit to carry in the body polynucleotide sequence that comprises SARS virus nucleocapsid polynucleotide sequence or its fragment. In one embodiment, described polynucleotides comprise SEQ ID N0:6051 or its fragment. The present invention also comprises the method for the immune response of trigger cell mediation, and the method comprises polynucleotides from its fragment to mammal that carry encoding SARS virus nucleocapsid albumen or.

The present invention includes the method for the inhibitor of screening SEQ ID NO:6051. Present invention resides in recombinant expressed SEQ ID NO:6051 in the host cell. The present invention includes the little molecule that can prevent that virus replication phase SEQ ID NO:6051 polypeptide is combined with the SARS viral RNA. The present invention includes the fusion that contains SEQ ID NO:6051. The protein positioning of the SEQ ID NO:6051 of prediction is as follows.

PSORT---protein positioning site estimation

Version 6.4 (WWW)

Species taxonomy: 4

* * inference step: 1

Primary Calculation ALOM (thresholding: 0.5)

Counting: 0

McG: detection signal peptide sequence (McGeoch)

UR length: 3

UR peak value: 0.19

CR net charge: 0

Distinguish scoring :-15.98

GvH: detection signal peptide sequence (von Heijne)

Signal peptide scoring (3.5) :-6.36

Possible cleavage site: 58

As if there is not a N-end signal sequence

The amino acid of the mature form of estimating forms:

Calculate 1 from the position

The ALOM New count: 0** territory electric charge is to-2

But in ALOM, detect cleavable signal peptide?: OB

ALOM: find cross-film district (Klein etc.)

Counting: 0 value: 5.04 thresholdings :-2.0

Peripheral possibility=5.04

The ALOM scoring of modifying :-1.91

Gavel: the border of detecting the target mtDNA sequence

Motif is positioned at: 17

    PRITFG

Distinguishing of target mtDNA sequence:

Negative (3.97)

* * inference step: 2

KDEL counting: 0

Verify the nonpolar signal peptide of sorting in the mitochondria

Mitochondrial matrix? scoring: 0.10

SKL motif (peroxisome signal peptide):

The position :-1 (399), counting: 0

Peroxisome tendentiousness amino acid forms: 0.04

The peroxidase body protein? state: unclear

AAC marks (peroxisome): 0.072

Lysosomal protein tendentiousness amino acid forms

Scoring: 0.96 state: unclear

Lysosomal modification is marked: 0.246

Examine the amount (nuclear) of alkaline residue

Examine 4 residue patterns of target nuclear

Find: position: 256 (4) KKPR

Find: position: 372 (5) KKKK

Examine 7 residue patterns of target nuclear

Examine Robbins ﹠ Dingwall consensus sequence (nuclear)

Find: position: 372 (3) KK KKTDEAQPLP QRQKK

Find: position: 373 (3) KK KTDEAQPLPQ RQKKQ

Final Robbins scoring (nuclear): 0.80

Examine RNA binding motif (nuclear or kytoplasm)

Nuclear is revised scoring: 0.90

Nuclear signal state: positive (0.90)

Examine the CaaX motif.

Examine the N-myristoylation.

Examine the CaaX motif.

-----final result-----

Nuclear---certainty=0.980 (sure)＜success 〉

Lysosome (chamber)---certainty=0.246 (sure)＜success 〉

Mitochondrial matrix gap---certainty=0.100 (sure)＜success 〉

Microbody (peroxisome)---certainty=0.072 (sure)＜success 〉

The N-glycosylation site of the SEQ ID NO:6051 of prediction is as follows through identifying.

The position possibility is agreed NGlyc result

48NNTA     0.6879         (9/9)    ++     (SEQ ID NO：7270)

270NVTQ    0.7684         (9/9)    +++    (SEQ ID NO：7271)

Residue numbering possibility threshold value suggestion

Thr        166        0.8547        0.6439        T

Thr        367        0.5575        0.5403        T

Thr        394        0.8217        0.5821        T

Therefore, the present invention includes the polypeptide of the amino acid sequence fragment that contains SEQ ID NO:6051, wherein said fragment comprises one or more N-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of one or more polypeptide that coding is identified above.

The present invention also comprises the polypeptide of the fragment that contains amino acid sequence SEQ ID NO:6051, and wherein said fragment does not contain one or more N-glycosylation sites of identifying above. The present invention also comprises the polynucleotides of this fragment of encoding.

In table 25, identified the T-epi-position of SEQ ID NO:6052. The present invention includes the polypeptide as antigen, wherein said polypeptide comprises: the amino acid sequence that (a) is selected from the T-epitope sequences that is accredited as SEQ ID NOS:9539-9752; (b) has the amino acid sequence of sequence homogeny with (a) amino acid sequence. The present invention also comprises the polynucleotide sequence of coding (a) or polypeptide (b). The present invention also comprises the method for expressing or carrying this polynucleotides by viral vectors and/or virion. The present invention also comprises the polypeptide of the T-epitope sequences that contains the one or more SEQ of being accredited as ID NOS:9539-9752, or the polynucleotides of this peptide species of encoding.

A variant that is included in the SEQ ID NO:6052 within the scope of the invention is SEQ ID NO:9964. Compare with SEQ ID NO:6052, the 54th residue Ile of this sequence replaced Thr.

Antigen preferably is used to: (1) is as the T-cellular antigens; (2) between I class MHC albumen (for example I class HLA) and described antigen fragment, form complex; (3) as the antigen of the immune response of trigger cell mediation; And/or (4) are as the antigen that causes CTL and reply. Disease and/or the infection that SARS virus causes can be prevented or treat to described purposes preferably. The invention provides polypeptide in the application of making the medicine that is used for the infection of immune mammal (especially people) opposing SARS virus, wherein said polypeptide as hereinbefore defined.

The invention provides the method that in mammal (especially people), causes immune response, the method comprises and gives the above step of defined polypeptide of described mammal, wherein said immune response is cell-mediated immune response, and preferred CTL replys. Described immune response is protectiveness or curative preferably.

The present invention includes the composition that contains SARS-NP or its fragment and contain SARS virus memebrane protein or its fragment. Described composition also can comprise one or more following adjuvants.

The present invention also comprises a kind of composition, comprising the polypeptide that contains SEQ ID NO:6051 or its fragment or have the sequence of sequence homogeny with it, and comprise the polypeptide that contains SEQ ID NO:6040 or its fragment or have the sequence of sequence homogeny with it. This composition can be used as, for example, and vaccine. This composition also can comprise one or more following adjuvants.

The present invention includes the composition that contains SARS-NP or its fragment and SARS virus spike protein or its fragment. In one embodiment, described nucleocapsid protein comprises the peptide sequence that contains SEQ ID NO:6051 or its fragment or have the sequence of sequence homogeny with it. In one embodiment, described spike protein comprises the polynucleotides that contain SEQ ID NO:6042 or its fragment or have the sequence of sequence homogeny with it. Described composition also can comprise one or more following adjuvants.

The present invention also comprises the composition that contains the SARS-NP specific antibody and contain SARS virus spike protein specific antibody. In one embodiment, described nucleocapsid protein specific antibody comprises the peptide sequence that contains SEQ ID NO:6051 or its fragment or have the sequence of sequence homogeny with it. In one embodiment, described spike protein specific antibody comprises the polynucleotides that contain SEQ ID NO:6042 or its fragment or have the sequence of sequence homogeny with it.

The present invention also comprises polynucleotide sequence and the fragment thereof that SARS virus is guarded in coronavirus, and its coded polypeptide. In contrast shown in Figure 7, identified this conserved sequence. This conserved sequence can be used for vaccine of the present invention or is used for diagnosticum of the present invention, kit and method.

The present invention also comprise SARS virus special with coronavirus in total polynucleotide sequence and fragment thereof. This SARS distinguished sequence is accredited as SEQ ID NOS:6040,6043,6044,6047,6048,6049 and 6050. This SARS distinguished sequence can be used for vaccine of the present invention or is used for diagnosticum of the present invention, kit and method.

The present invention also comprise can be used as in diagnosticum, the kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have in the method for SARS virus polynucleotide sequence as probe or primer. Comprise in this polynucleotide sequence that the present invention includes among the SEQ ID NOS:6076-6265 through being accredited as one or more primer sequences (table 5). The present invention also comprises and containing among the SEQ ID NOS:6076-6265 through being accredited as the polynucleotide sequence of one or more primer sequence complement.

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the probe of method of SARS virus or the polynucleotide sequence of primer. Comprise in this polynucleotide sequence that the present invention includes among the SEQ ID NOS:6266-6343 through being accredited as one or more primer sequences (table 6). The present invention also comprises the polynucleotide sequence that contains the complement through being accredited as one or more primer sequences among the SEQ ID NOS:6266-6343.

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the probe of method of SARS virus or the polynucleotide sequence of primer. Comprise in this polynucleotide sequence that the present invention includes among the SEQ ID NOS:6344-6392 through being accredited as one or more primer sequences (table 7). The present invention also comprises the polynucleotide sequence that contains the complement through being accredited as one or more primer sequences among the SEQ ID NOS:6344-6392.

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the probe of method of SARS virus or the polynucleotide sequence of primer. Comprise in this polynucleotide sequence that the present invention includes among the SEQ ID NOS:6393-6559 through being accredited as one or more primer sequences (table 8 and table 9). The present invention also comprises the polynucleotide sequence that contains the complement through being accredited as one or more primer sequences among the SEQ ID NOS:6393-6559.

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the probe of method of SARS virus or the polynucleotide sequence of primer. Comprise in this polynucleotide sequence that the present invention includes among the SEQ ID NOS:6560-6568 through being accredited as one or more primers and probe sequence. The present invention also comprises the polynucleotide sequence that contains the complement through being accredited as one or more primer sequences among the SEQ ID NOS:6560-6568.

The present invention includes the peptide sequence that contains arbitrary even-numbered sequence among the SEQ ID NOS:7272-7290 or its fragment or have the sequence of sequence homogeny with it. The present invention also comprises arbitrary even-numbered sequence or its fragment among the coding SEQ ID NOS:7272-7290 or has the sequence polynucleotide sequence of sequence homogeny with it. The example of this polynucleotide sequence is the sequence of the odd-numbered among the SEQ ID NOS:7273-7291.

The present invention includes the polynucleotide sequence that contains the common intergenic sequence of each ORF of SARS virus. Think that SARS virus utilizes this sequence to translate ORF as signal. Described intergenic sequence comprises 10 polymers SEQ ID NO:7292, or the optional hexamer SEQ ID NO:7293 that comprises. When virus when just (+) RNA chain is being transcribed into (-) RNA chain with it, virus structure utilization (-) the chain template that is copying is transcribed the before nucleotides of 5 ' end of first intergenic sequence, then then sequence between open gene transcribes selected ORF again. Then virus produce a plurality of mRNA that comprise this 5 ' end, intergenic sequence and coded sequence. For the more details of understanding nest the class of virus virus replication (comprising coronavirus) can referring to, for example, Ziebuhr etc., " protease of encoding viral and proteolysis process in the nest the class of virus virus " (Virus-encoded proteinases and proteolytic processing in the Nidovirales), Journal of General Virology 81:853-879 (2000) fits into this paper as a reference in it.

The present invention includes the polynucleotide sequence that contains SEQ ID NO:7292 or its complement. The present invention includes the polynucleotide sequence that contains SEQ ID NO:7293 or its complement. The present invention also comprises and contains SARS virus genome 5 ' terminal nucleotide or its reverse complemental thing, and comprises the polynucleotide sequence of intergenic sequence or its reverse complemental thing. These polynucleotides also can comprise one or more SARS virus ORFs. The example of polynucleotide sequence that comprises SARS virus genome 5 ' end and then be the nucleotides of intergenic sequence is SEQ ID NOS:7294-7301. The present invention includes polynucleotide sequence, wherein comprise and be selected from lower group sequence: SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301, or its fragment, or has the sequence of sequence homogeny with it. In one embodiment, described polynucleotides are imperfect to be made of known SARS virus sequence.

The SARS virus intergenic sequence can be used to produce the RNAi molecule. This SARS virus specific RNA i molecule can be used to treat SARS virus and infects. The present invention includes the RNAi molecule that contains double stranded rna molecule, a RNA chain comprises and is selected from lower group sequence in this two strands: SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301, or its fragment. Preferred described RNA chain contains the sequence that is selected from SEQ ID NO:7292 and SEQ ID NO:7293. Preferred another RNA chain contains the reverse complemental thing of article one chain or the polynucleotide sequence of hybridizing with energy article one chain.

Utilize in the method for RNAi treatment SARS virus infection in the present invention, comprise the si RNA molecule that gives the mammal effective dose. Preferred described RNAi molecule comprises above-mentioned molecule. This specification IV part has further been discussed the application of intergenic sequence aspect RNAi.

The present invention also comprise the antisense sequences of SARS virus antisense base sequences-preferred SARS virus intergenic sequence-application. This antisense sequences can be used to treat the individuality that infected by SARS virus. Can design the antisense sequences of the SARS virus intergenic sequence that can be combined with the SARS virus polynucleotides to stop virus replication mechanism to enter this intergenic sequence. This antisense sequences also can be used for whether existing in the identification of organism sample SARS virus. Antisense sequences self can be labeled, and perhaps can detect the antisense sequences of being combined with viral polynucleotides with the known method in this field. The antisensenucleic acids of design can with the RNA specific binding, this will cause forming RNA-DNA or RNA-RNA crossbred, thereby stop the translation of dna replication dna, reverse transcription or mRNA. Can hinder the expression of corresponding gene based on the antisense polynucleotides of selected sequence. Antisense polynucleotides in connection with and/or hinder the translation of corresponding mRNA.

The present invention also comprises the application with the intergenic region of ribozyme.

Trans cutting catalytic RNA (ribozyme) is the RNA molecule with endoribonuclease activity. Ribozyme is custom-designed for particular target, and the target mRNA must contain specific nucleotide sequence. They can be modified and can specificity cut site in the cell RNA main chain of the RNA site of any kind-especially through genetic engineering. This cutting makes mRNA become unstable and stops protein expression. Importantly, ribozyme can be used to suppress the expression of unknown function gene, thereby can determine the function of this gene in external or body by detecting phenotypic effect.

A kind of ribozyme motif commonly used is hammerhead ribozyme, this ribozyme to the substrate sequence require minimum. The design description of hammerhead ribozyme is at Usman etc., Current Opifz.Struct.Biol. (1996) 6:527-533. Usman has also discussed the therapeutical uses of ribozyme. Also can prepare and use ribozyme: Long etc., FASEB J. (1993) 7:25 according to following description; Symons, Ann.Rev.Biochem. (1992) 61:641; Perrotta etc., Biochem. (1992) 31:16-17; Ojwang etc., Proc.Natl.Acad.Sci. (USA) (1992) 89:10802-10806; With United States Patent (USP) 5254678. United States Patent (USP) 5144019 has been described the cutting of ribozyme to HIV-I RNA; United States Patent (USP) 5116742 has been described the method with ribozyme cutting RNA; United States Patent (USP) 5225337 and Koizumi etc., Nucleid Acid Res. (1989) 17:7059-7071 has described the specific method of raising ribozyme. Koizumi etc., Nucleid Acid Res. (1989) 17:7059-7071 has also described preparation and the use of the ribozyme fragment of hammerhead structure. Chowrira and Burke, Nucleid Acid Res. (1992) 20:2835 has described preparation and the use of the ribozyme fragment of hairpin structure. Also can transcribe the manufacturing ribozyme by rolling ring, such as Daubendiek ﹠ Kool, Nat. Biotechnol. (1997) 15 (3): 273-277 is described.

Can be modified or be made into branched structure in the hybridization zone of ribozyme, such as Horn Urdea, Nucleid Acid Res. (1989) 17:6959-67 is described. The method that also can be familiar with the technical staff who is proficient in this field changes the foundation structure of ribozyme by chemical process, and the ribozyme of chemical synthesis is used as the synthetic oligonucleotide derivative of modifying with monomeric unit. In treatment, the conveying of liposome-mediated ribozyme has improved cellular uptake, and such as Birikh etc., Eur.J.Biochem. (1997) 245:1-16 is described.

The therapeutic of ribozyme and functioning gene group are used and are started from wanting the understanding of repressed gene coded sequence part. In the present invention, target sequence preferably includes the intergenic sequence of SARS virus. Preferred described sequence is selected from SEQ ID NO:7292 and SEQ ID NO:7293. The target cleavage site is selected from target sequence, and classifies the basis as with 5 ' and 3 ' nucleotides sequence of cleavage site both sides and make up ribozyme. Preferred 5 ' nucleotide sequence comprises the 5 ' non-translational region of SARS virus. Then can make up ribozyme from one or more polynucleotide sequences that are selected from lower group: SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301.

Described the antisense therapy that HIV infects in the following list of references, these documents are included in as a reference in full. (mRNA of antisense RNA and gag, tat, rev, env is complementary) (Sezakiel etc., 1991, J.Virol.65:468-472; Chatterjee etc., 1992, Science 258:1485-1488; Rhodes etc., 1990, J.Gen.Virol.71:1965; Rhodes etc., 1991, AIDS 5:145-151; Sezakiel etc., 1992, J.Virol.66:5576-5581; Joshi etc., 1991, J.Virol.65:5524-5530).

The present invention includes and utilize decay RNA to destroy copying of SARS virus and life cycle. Making and utilizing the method for this decay RNA treatment virus infections is that this field is known. The present invention includes to the gene of infected cell delivery coding (for example) SARS virus intergenic sequence. Preferred described sequence comprises one or more sequences that are selected from lower group: SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301. Preferred described sequence comprises one or more sequences that are selected from lower group: SEQ ID NO:7292 and SEQ ID NO:7293. Preferred described sequence comprises SEQ ID NO:7293.

In the present invention, but carry with the translation process of the disjunct intergenic sequence break virus of SARS virus ORF RNA and reduce virus protein and produce. The similar approach for the treatment of HIV virus infections has been described. Narrate the decay RNA that utilizes HIV TAR or RRE below with reference to data and treated the HIV infection. Fit into this paper in these references as a reference. (Sullenger etc., 1990, Cell 63:601-608; Sullenger etc., 1991, J.Virol.65:6811-6816; Lisziewicz etc., 1993, New Biol.3:82-89; Lee etc., 1994, J.Virol. 68:8254-8264), ribozyme (Sarver etc., 1990, Science 247:1222-1225; Wecrasinghe etc., 1991, J.Virol.65:5531-5534; Dropulic etc., 1992, J.Virol.66:1432-1441; Ojwang etc., 1992, Proc.Natl.Acad.Sci.USA.89:10802-10806; Yu etc., 1993, Proc.Natl.Acad.Sci.USA.90:6340-6344; Yu etc., 1995, Proc.Natl.Acad.Sci.USA.92:699-703; Yamada etc., 1994, Gene Therapy 1:38-45).

The present invention includes the SARS virus intergenic sequence at diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have application in the method for SARS virus. This diagnosticum, kit and method will further be narrated in the specification part ii.

The primer pair of SARS polynucleotide sequence the present invention includes to increase, comprising (i) the first primer, wherein comprise and the essentially identical sequence of the Sequence that is selected from lower group: SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301, and (ii) the second primer, wherein comprise and the basic complementary sequence of a part of sequence that is selected from sequence SEQ ID NO:1 and sequence SEQ ID NO:2, like this, primer has just been determined a template sequence to (i) with (ii) in the sequence of sequence SEQ ID NO:1 and sequence SEQ ID NO:2 formation. Preferably (i) first primer comprises and the essentially identical sequence of a part of sequence that is selected from SEQ ID NO:7292 and SEQ ID NO:7293. Preferably (i) first primer comprises the essentially identical sequence of a part of sequence with SEQ ID NO:7293. The length of the amplicon that described the first and second primers are determined is preferably between 50 and 250 nucleotides. These primers can be chosen wantonly in addition, and mark is beneficial to its detection. Being used for the method and composition of labeled primer will further discuss in the application's III part.

The present invention's also comprise increasing primer pair of SARS polynucleotide sequence, comprising (i) the first primer, wherein comprise and the essentially identical sequence of complement part that is selected from lower group Sequence: SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301, and (ii) the second primer, wherein comprise and the basic complementary sequence of a part that is selected from sequence SEQ ID NO:1 and sequence SEQ ID NO:2 complement, like this, primer is to just having determined a template sequence in the sequence that is made of sequence SEQ ID NO:1 and sequence SEQ ID NO:2. The length of the amplicon of being determined by described the first and second primers is preferably between 50 and 250 nucleotides. Primer can be chosen wantonly and be labeled so that detect. Being used for the method and composition of labeled primer will further discuss in the application's III part.

The present invention includes a kit, comprising (i) the first primer, wherein comprise and the essentially identical sequence of the Sequence that is selected from lower group: SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301, and (ii) the second primer, wherein comprise and the basic complementary sequence of a part that is selected from sequence SEQ ID NO:1 and sequence SEQ ID NO:2, like this, primer has just been determined a template sequence to (i) with (ii) in the sequence that is made of sequence SEQ ID NO:1 and sequence SEQ ID NO:2. Preferably (i) first primer comprises the essentially identical sequence of a part with the sequence that is selected from SEQ ID NO:7292 and SEQ ID NO:7293. Preferably (i) first primer comprises the essentially identical sequence of a part with the sequence of SEQ ID NO:7293. Primer can be chosen wantonly and be labeled so that detect. Being used for the method and composition of labeled primer will further discuss in the application's III part.

Other preferred kit comprises (i) first primer, wherein comprise and the essentially identical sequence of complement part of a part that is selected from lower group sequence: SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301, and (ii) the second primer, wherein comprise and the basic complementary sequence of complement part that is selected from sequence SEQ ID NO:1 and sequence SEQ ID NO:2, like this, primer is to just having determined a template sequence in the sequence that is made of sequence SEQ ID NO:1 and sequence SEQ ID NO:2.

The present invention also comprises the attenuation SARS virus as vaccine, and the expression of virus structural protein or non-structural protein reduces thereby the intergenic region in this virus has suddenlyd change. This attenuation SARS virus can have one or more interpolations, disappearance or insertion in virus genomic one or more intergenic regions. Preferred this attenuation SARS virus has one or more interpolations, disappearance or insertion in the sequence that is selected from SEQ ID NO:7292 and SEQ ID NO:7293. Preferably in SEQ ID NO:7293, one or more interpolations, disappearance or insertion are arranged.

The present invention also comprises can suppress the little molecule that SARS virus duplicating mechanism such as ribonucleoprotein are combined or are associated with the viral genome intergenic region. Preferred described little molecular energy suppresses SARS virus mechanism is combined with the sequence that is selected from SEQ ID NO:7292 and SEQ ID NO:7293 or associates. Preferred described little molecular energy suppresses SARS virus mechanism is combined with SEQ ID NO:7293 or associates. The present invention comprises that also screening is used for the treatment of the micromolecular method that SARS virus infects, and described method comprises and adopts a kind of assay method to identify the little molecule that disturbs the SARS virus duplicating mechanism to combine with the genomic intergenic region of SARS virus.

The present invention also provides a kind of new SARS polynucleotide sequence SEQ ID NO:9968. 6 reading frames of all of the sequence of this 690mer are shown in Figure 113. In the composition amino acid sequence of Figure 113 at least 4 amino acid are arranged, they are used as SEQ ID NOS:9969-10032 and list.

Therefore, the present invention includes the polynucleotide sequence that contains SEQ ID NO:9968. The present invention also provides the polynucleotide sequence that has the sequence homogeny with SEQ ID NO:9968. Sequence homogeny degree is preferably greater than 50% (for example, 60%, 70%, 80%, 85%, 88%, 90%, 92%, 95%, 99% or higher).

The present invention includes the amino acid sequence by the polynucleotide sequence coding of SEQ ID NO:9968, comprise the amino acid sequence that is selected from SEQ ID NOs:9969-10032. Preferred described amino acid sequence comprises SEQ ID NO:9997 or comprises SEQ ID NO:9998.

The present invention also provides the amino acid sequence that has the sequence homogeny with the amino acid sequence of SEQ ID NO:9968 coding. The invention provides the amino acid that has the sequence homogeny with the amino acid sequence that is selected from SEQ ID NOS:9969-10032. Sequence homogeny degree is preferably greater than 50% (for example, 60%, 70%, 80%, 85%, 88%, 90%, 92%, 95%, 99% or higher).

The part of SEQ ID NO:9968 is complementary with the SARS polynucleotide sequence that is commonly called " BNI-1 " (SEQ ID NO:10033) of before having delivered with about 98% homogeny. BNI-1 is sequenced in national tropical infectious disease reference center (National Reference Center for Tropical Infectious Diseases) the tropical medicine Bernhard Nocht research institute of Hamburg, Germany. The BNI-1 sequence is published at WHO website http://www.who.int/csr/sars/primers/en on April 4th, 2003, and be published in Dorsten etc., " identify the novel coronavirus in the severe acute respiratory syndrome patient " (Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome), New England Journal of Medicine, on April 10th, 2003 online publishing in http://www.nejm.org. These two parts of references are all included in this paper as a reference. 6 reading frames of this 302mer sequence are shown in Figure 114 (also can referring to Figure 129). In the composition amino acid sequence of Figure 114 at least 4 amino acid are arranged, they are used as SEQ ID NOS:10034-10065 and list. The contrast of SEQ ID NO:10034 and SEQ ID NO:9997 is shown in Figure 130.

The invention provides the polynucleotide sequence that comprises SEQ ID NO:9968 fragment. In one embodiment, this fragment is not that complete sequence by SEQ ID NO:10033 or known coronavirus consists of.

The invention provides the amino acid sequence of the fragment that comprises the amino acid sequence of being encoded by SEQ ID NO:9968. In one embodiment, described fragment is not to be made of the amino acid sequence of SEQ ID NO:10033 coding or the sequence of known coronavirus fully.

The invention provides the amino acid sequence of the fragment that comprises the amino acid sequence that is selected from SEQ ID NOS:9969-10032. In one embodiment, described fragment is not to be made of the amino acid sequence of the polynucleotide sequence coding of SEQ ID NO:10033 or the sequence of known coronavirus fully.

5 ' the end of SEQ ID NO:9968 has 100 nucleotides not mate with any part of BNI-1 polynucleotide sequence (SEQ ID NO:10033) approximately. This unmatched part rows is in SEQ ID NO:10066. Therefore, the present invention also provides and has comprised the polynucleotides that contain SEQ ID NO:10066 sequence, has the polynucleotide sequence of sequence homogeny with SEQ ID NO:10066 or contain the polynucleotide sequence of the fragment of SEQ ID NO:10066.

The present invention also comprises the amino acid sequence by SEQ ID NO:10066 coding, have the amino acid sequence of sequence homogeny with the amino acid sequence of SEQ ID NO:10066 coding, or comprise the amino acid sequence by the amino acid sequence fragment of SEQ ID NO:10066 coding. Preferred described amino acid sequence comprises SEQ ID NO:10067.

SEQ ID NO:9997/9998 is proved the pol1ab zone homology with several coronavirus. Figure 115 has shown the amino acid sequence (SEQ ID NO:10068) of SEQ ID NOS:9997/9998 and bovine coronavirus pol 1ab. The contrast of the amino acid sequence (SEQ ID NO:10070) of the amino acid sequence of avian infectious bronchitis virus pol 1ab (SEQ ID NO:10069) and murine hepatitis virus pol 1ab. The consensus amino acid sequences of SEQ ID NOS:9997/9998, SEQ ID NO:10068, SEQ ID NO:10069 and SEQ ID NO:10070 is presented at last column (for example SEQ ID NO:10071) of Figure 115 contrast.

Shown in Figure 113, the polynucleotide sequence of the SEQ ID NO:9997 of coding has a terminator codon after codon 205, between the SEQ ID NOS:9997 and 9998. This terminator codon can be chosen wantonly and be removed and make this amino acid sequence continuously (SEQ ID NO:10072). Therefore, the invention provides and contain SEQ ID NO:9997 and/or SEQ ID NO:9998, or SEQ ID NO:10072, and comprise the amino acid sequence of the amino acid sequence of coding coronavirus pol1ab gene or its fragment C-end.

Shown in Figure 115, SEQ ID NOS:10068,10069,10070 and 10071 is before the amino acid that SEQ ID NO:9997 contains is positioned at the N-end. Amino acid sequence provided by the invention also contains SEQ ID NO:9997 and comprises the amino acid sequence of coding coronavirus pol1ab albumen or its fragment N-end.

The pol1ab sequence of Figure 115 comprises a code area, and this zone represents with " * " in Figure 117. In Figure 115, use and pass the starting point that consensus sequence SEQ ID NO:10071 amino acid 6080 arrow before represents this genome area. The terminal point of this genome area represents with passing this consensus sequence amino acid 6604 arrow before. Amino acid sequence provided by the invention contains SEQ ID NO:9997 and/or SEQ IID NO:9998, or SEQ ID NO:10072, and comprise described SEQ ID NO:9997 and/or SEQ ID NO:9998, or the first amino acid sequence before the SEQ ID NO:10072N-end, the N-end sequence of wherein said first amino acid sequence and known coronavirus pol1ab " * " albumen or its fragment has homology.

The present invention also provides the amino acid sequence that contains SEQ ID NO:9997 and SEQ ID NO:9998, wherein, terminator codon after the SEQ ID NO:9971 is removed (being SEQ ID NO:10072), and comprise the second amino acid sequence after the SEQ ID NO:9998C end, the C end homology of wherein said the second amino acid sequence and known coronavirus pol1ab " * " albumen or its fragment.

The example of this protein relatively illustrates in Figure 118, and they are SEQ ID NOS:10073-10077. SEQ ID NO:10073 comprises SEQ ID NO:9997, and comprises before avian infectious bronchitis virus pol1ab " * " the albumen N-end and the amino acid after the C-end. SEQ ID NO:10074 comprises SEQ ID NO:9997, and comprises before bovine coronavirus pol1ab " * " the albumen N-end and the amino acid after the C-end. SEQ ID NO:10075 comprises SEQ ID NO:9997, and comprises before murine hepatitis virus pol1ab " * " the albumen N-end and the amino acid after the C-end. SEQ ID NO:10076 comprises SEQ ID NO:9997, and comprises before avian infectious bronchitis virus, bovine coronavirus and murine hepatitis virus pol1ab " * " the albumen consensus sequence N-end and the amino acid (Figure 115) after the C-end. SEQ ID NO:10077 comprises the consensus sequence of SEQ ID NOS:10073-10076.

The present invention includes and be selected from SEQ ID NOS:10073,10074,10075,10076 and 10077 amino acid sequence. The present invention also comprises and contains the amino acid sequence that is selected from SEQ ID NOS:10073,10074,10075,10076 and 10077 amino acid sequence fragment. The present invention also comprises and the amino acid sequence that is selected from SEQ ID NOS:10073,10074,10075,10076 and 10077 amino acid sequence and has the sequence homogeny.

The present invention includes the polynucleotides that coding is selected from SEQ ID NOS:10073,10074,10075,10076 and 10077 amino acid sequence. The present invention includes the polynucleotides that have the sequence homogeny with the polynucleotides that are selected from SEQ ID NOS:10073,10074,10075,10076 and 10077 amino acid sequence of encoding. The present invention includes the fragment of coding SEQ ID NOS:10073,10074,10075,10076 and 10077 polynucleotides.

Shown in Figure 113, SEQ ID NO:9968 comprises the sequence of coding SEQ ID NO:10020, the terminator codon after it, terminal threonine (Thr) residue of given C-. The corresponding sequence of the amino acid sequence of BNI-1 coding is SEQ ID N0:10078, and its continuity is until the C-of SEQ ID NO:10020 is terminal. Therefore, the present invention includes and contain amino acid sequence SEQ ID NO:10020 or have the amino acid sequence of sequence homogeny with SEQ ID NO:10020 or contain the protein of amino acid sequence of the fragment of SEQ ID NO:10020, wherein, the C-terminal residue of described protein is threonine. The C-end of preferred described protein is-ST. More preferably the C-end of described protein is-EST. The present invention also comprises and contains amino acid sequence SEQ ID NO:10078 or have the amino acid sequence of sequence homogeny with SEQ ID NO:10078, or contain the protein of amino acid sequence of the fragment of SEQ ID NO:10078, wherein, the C-terminal residue of described protein is Thr. The C-end of preferred described protein is-ST. More preferably the C-end of described protein is-EST.

The SEQ ID NO:9968 54mer amino acid sequence SEQ ID NO:10015 that also encodes. The polynucleotides of this coding SEQ ID NO:10015 have two terminator codons (Figure 113) at its C-end. The respective regions of BNI-1 sequence does not contain this 54mer sequence. Therefore, the present invention includes and contain amino acid sequence SEQ ID NO:10015 or have the amino acid sequence of sequence homogeny with SEQ ID NO:100015 or contain the protein of amino acid sequence of the fragment of SEQ ID NO:10015. The present invention also comprises and contains SEQ ID NO:10015 and contain the before polypeptide of the first amino acid sequence of SEQ ID NO:10015N-end.

SEQ ID NO:9968 encoding amino acid sequence SEQ ID NO:9969. This polynucleotide sequence contains a terminator codon at the C-end of SEQ ID NO:9969. Therefore, the present invention includes and contain amino acid sequence SEQ ID NO:9969 or have the protein of the amino acid sequence of sequence homogeny with SEQ ID NO:9969. The present invention also comprises the polypeptide that contains SEQ ID NO:9969 and contain SEQ ID NO:9969N-end first amino acid sequence before. The present invention also comprises the polypeptide that contains sequence SEQ ID NO:10079.

SEQ ID NO:9968 encoding amino acid sequence QRT (Figure 113) is thereafter a terminator codon. Therefore, the present invention includes the protein that contains amino acid sequence QRT. The present invention also comprises and contains amino acid sequence QRT and contain the before polypeptide of the first amino acid sequence of sequence QRT N-end.

SEQ ID NO:9968 encoding amino acid sequence SEQ ID NO:10022 is the terminator codon of its C-end subsequently. Therefore, the present invention includes and contain amino acid sequence SEQ ID NO:10022 or have the protein of the amino acid sequence of sequence homogeny with SEQ ID NO:10022. The present invention also comprises and contains SEQ ID NO:10022 and contain the before polypeptide of the first amino acid sequence of SEQ ID NO:10022N-end.

SEQ ID NO:9968 encoding amino acid sequence SEQ ID NO:10027. In the SEQ ID NO:10027 coded sequence at least 3 initiation codons are arranged, in Figure 119, represent with underscore. The ORF that first initiation codon is pointed out is SEQ ID NO:10081. The ORF that second initiation codon pointed out is SEQ ID NO:10082. The 3rd ORF that initiation codon is pointed out is SEQ ID NO:10083.

The invention provides a kind of new SARS polynucleotide sequence SEQ ID NO:10084. 6 reading frames of all of this 1463mer sequence are shown in Figure 120 (also can referring to Figure 122). In the composition amino acid sequence of Figure 120 at least 4 amino acid are arranged, they are used as SEQ ID NOS:10085-10209 and list (referring to Figure 120 A-120F).

The present invention includes the polynucleotide sequence that contains SEQ ID NO:10084. The present invention also provides the polynucleotide sequence that has the sequence homogeny with SEQ ID NO:10084. The present invention also provides the polynucleotide sequence of the fragment that contains SEQ ID NO:10084. In one embodiment, this polynucleotide passage is not to be made of SEQ ID NO:10033 or known coronavirus polynucleotide sequence or known SARS polynucleotide sequence fully.

The present invention includes the amino acid sequence by the polynucleotide sequence coding of SEQ ID NO:10084, comprise the amino acid sequence of Figure 120 A-120F, for example be selected from the sequence of SEQ ID NOS:10085-10209. Preferred described amino acid sequence comprises SEQID NO:10149.

The present invention also provides the amino acid sequence that has the sequence homogeny with the amino acid sequence of SEQ ID NO:10084 coding. The invention provides the amino acid that has the sequence homogeny with the amino acid sequence of Figure 120 A-120F, for example be selected from the sequence of SEQ ID NOS:10085-10209.

The present invention also provides the fragment of the amino acid sequence of SEQ ID NO:10084 coding. The present invention also provides the fragment of the amino acid sequence that is selected from SEQ ID NOS:10085-10209. In one embodiment, this fragment is not to be made of the amino acid sequence of SEQ ID NO:10033 coding or the amino acid sequence of known coronavirus or the amino acid sequence of known SARS virus fully. SEQ ID NO:10033 and SEQ ID NO:10084 be complementary the part contrast be shown in Figure 121.

In one embodiment, the present invention includes the amino acid sequence that contains SEQ ID NO:10149. The SEQ ID NO:10149 of polynucleotide sequence SEQ ID NO:10084 and coding relatively is shown in Figure 122 (5 ' 3 ' frame 3). Analyze 5 ' 3 ' frames, 3 translation things (Figure 123) by the computer program (NetStart 1.0) of prediction methionine initiation codon and shown SEQ ID NOS:10210-10215.

The present invention includes and contain the protein that is selected from SEQ ID NO:10210, SEQ ID NO:10211, SEQ ID NO:10212, SEQ ID NO:10213, SEQ ID NO:10214 and SEQ ID NO:10215 amino acid sequence. The present invention includes and the protein that is selected from SEQ ID NO:10210, SEQ ID NO:10211, SEQ ID NO:10212, SEQ ID NO:10213, SEQ ID NO:10214 and SEQ ID NO:10215 amino acid sequence and has the sequence homogeny. In one embodiment, this protein is not that complete amino acid sequence by known SARS virus or known coronavirus consists of.

The present invention includes the fragment that contains the protein that is selected from SEQ ID NO:1021O, SEQ ID NO:10211, SEQ ID NO:10212, SEQ ID NO:10213, SEQ ID NO:10214 and SEQ ID NO:10215 amino acid sequence. In one embodiment, this fragment is not that complete amino acid sequence by known SARS virus or known coronavirus consists of.

In one embodiment, the present invention includes the polypeptide that contains the amino acid sequence that is selected from SEQ ID NO:10210, SEQ ID NO:10211 and SEQ ID NO:10212. Figure 124 comprises the partial results of GenBank being carried out the BLAST algorithm of SEQ ID NO:10210. These results show, SEQ ID NOS:10210,10211 and 10212 and the RNA polymerase of coronavirus RNA polymerase, especially murine hepatitis virus, bovine coronavirus and bird infective bronchitis similar on function.

In one embodiment, the present invention relates to contain the polypeptide of the second amino acid sequence of the first amino acid sequence of being selected from SEQ ID NO:10210, SEQ ID NO:10211 and SEQ ID NO:10212 and coronavirus ORF1ab sequence C-end. Preferred this second amino acid sequence is from bovine coronavirus. An example of this embodiment is SEQ ID NO:10216. 1-481 the amino acid of SEQ ID NO:10216 is the first amino acid sequence of SEQ ID NO:10210, and the second amino acid sequence (Gi 26008080) that 482-1152 amino acid is bovine coronavirus orf1ab polyprotein C-end is (SEQ ID NO:10217) (NP_150073.2).

Therefore, the present invention includes the polypeptide that contains SEQ ID NO:10216. The present invention also comprises the polypeptide of the second amino acid sequence of the first amino acid sequence of containing SEQ ID NO:10210 and SEQ ID NO:10217. The present invention also comprise contain with SEQ ID NO:10210 homogeny greater than the first amino acid sequence of x% and with the polypeptide of SEQ ID NO:10217 homogeny greater than the second amino acid sequence of y%, wherein, x more than or equal to 85% (for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher), y is more than or equal to 60% (for example, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher).

The present invention also comprises the polypeptide of the fragment that contains SEQ ID NO:10210, and wherein said fragment contains an epi-position. Use the 17mer window, the epi-position of the SEQ ID NO:10210 by computer forecast is shown in Figure 125 A (Hopp﹠ Woods) and Figure 125 B (Kyte ﹠ Doolittle).

The amino acid sequence of SEQ ID NO:10210 is also at amino acid 81-84 (NNTE; SEQ ID NO:10218) and 180-183 (NHSV; SEQ ID NO:10219) glycosylation site that contains two predictions on. Therefore, the present invention includes the polypeptide of the fragment that contains SEQ ID NO:10210, wherein said fragment contains glycosylation site. The present invention also comprises the polypeptide of the fragment that contains SEQ ID NO:10210, and wherein said fragment has Asn at the 81st. Preferred this Asn is by glycosylated. The present invention also comprises the polypeptide of the fragment that contains SEQ ID NO:10210, and wherein said fragment has Asn at the 180th. Preferred this Asn is by glycosylated.

In one embodiment, the present invention includes and contain from the amino acid sequence among Figure 120 D and/or from the polypeptide of the amino acid sequence of SEQ ID NOS:10150-10160 (for example from SEQ ID NOS:10154,10155,10158 and 10160 amino acid sequence). Contain the amino acid sequence that starts from Met and end at a terminator codon through evaluation among the SEQ ID NO:10154: SEQ ID NOS:10220-10227.

Therefore, the present invention includes and contain the amino acid sequence that is selected from SEQ ID NO:10220, SEQ ID NO:10221, SEQ ID NO:10222, SEQ ID NO:10223, SEQ ID NO:10224, SEQ ID NO:10225, SEQ ID NO:10226 and SEQ ID NO:10227, or its fragment or have the polypeptide of the amino acid sequence of sequence homogeny with it.

In one embodiment, the present invention includes the amino acid sequence that contains among Figure 120 E, for example from SEQ ID NOS:10161-10182, the polypeptide of SEQ ID NOS:10171 and 10176 amino acid sequence especially. In SEQ ID NOS:10171-10176, can identify the following Met of starting from and end at the amino acid sequence of a terminator codon: SEQ ID NO:10228 and SEQ ID NO:10229.

Therefore, the present invention includes and contain the amino acid sequence that is selected from SEQ ID NO:10228 and SEQ ID NO:10229, or its fragment or have the polypeptide of the amino acid sequence of sequence homogeny with it.

In one embodiment, the present invention includes the polypeptide of the amino acid sequence (for example SEQ ID NOS:10183-10209) that contains Figure 120 F. In Figure 120 F, can identify the following Met of starting from and end at the amino acid sequence of a terminator codon: SEQ ID NO:10187. Therefore, the present invention includes amino acid sequence or its fragment that contains SEQ ID NO:10187 or the polypeptide that has the amino acid sequence of sequence homogeny with it.

In one embodiment, polynucleotides of the present invention do not contain one of following primer, and these primers are disclosed in http://content.nejnz.org/cgi/reprint/NEJMoa030781v2.pdf:

5’GGGTTGGGACTATCCTAAGTGTGA3’(SEQ ID NO：10230)

5’TAACACACAACICCATCATCA3’(SEQ ID NO：10231)

5’CTAACATGCTTAGGATAATGG3’(SEQ ID NO：10232)

5’GCCTCTCTTGTTCTTGCTCGC3’(SEQ ID NO：10233)

5’CAGGTAAGCGTAAAACTCATC3’(SEQ ID NO：10234)

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the polynucleotide sequence of probe of the method for SARS virus. The present invention includes the polynucleotide primers (SEQ ID NOS:10235-10258) that table 31 is identified, forward primer SEQ ID NOS:10259-10281 and reverse primer SEQ ID NOS:10282-10298. The present invention also comprises the polynucleotide sequence with the arbitrary primer sequence complementation that discloses here.

The invention provides SARS polynucleotide sequence SEQ ID NO:10299. Figure 126 comprises all 6 reading frames (also can referring to Figure 131) of this sequence. In the composition amino acid sequence of Figure 126 at least 4 amino acid are arranged, they are used as SEQ ID NOS:10300-10337 and list.

Therefore, the present invention includes the polynucleotide sequence that contains SEQ ID NO:10299. The present invention also provides the polynucleotide sequence that has the sequence homogeny with SEQ ID NO:10299. The present invention also provides the polynucleotide sequence that contains SEQ ID NO:10299 fragment. In one embodiment, this polynucleotide passage is not to be made of the known polynucleotide sequence of SARS virus or the known polynucleotide sequence of coronavirus fully.

The present invention includes the amino acid sequence by the polynucleotide sequence coding of SEQ ID NO:10299, comprise the amino acid sequence shown in Figure 126, and the amino acid sequence that is selected from SEQ ID NOS:10300-10337. Preferred described amino acid sequence comprises SEQ ID NO:10316.

The present invention also provides the amino acid sequence that has the sequence homogeny with the amino acid sequence by SEQ ID NO:10299 coding. The invention provides the amino acid sequence that has the sequence homogeny with the amino acid sequence that is selected from SEQ ID NO:10300-10337.

The present invention also provides the fragment by the coded amino acid sequence of SEQ ID NO:10299. The present invention also provides the fragment of the amino acid sequence that is selected from SEQ ID NOS:10300-10337. In one embodiment, this fragment is not to be made of the known amino acid sequence of SARS virus or the known amino acid sequence of coronavirus fully.

In one embodiment, the present invention includes the amino acid sequence that contains SEQ ID NO:10316. Coding ORF in the SEQ ID NO:10316 comprises SEQ ID NO:10338 and SEQ ID NO:10339.

In one embodiment, the present invention includes the amino acid sequence that contains from the sequence of SEQ ID NO:10299 5 ' 3 ' frames 1 translation thing. In this translation thing, found the ORF of following coding: SEQ ID NO:10340.

In one embodiment, the present invention includes the amino acid sequence that contains from the sequence of SEQ ID NO:102995 ' 3 ' frame 1 translation thing. In this translation thing, found the ORF of following coding: SEQ ID NO:10341.

In one embodiment, the present invention includes the amino acid sequence that contains from the sequence of SEQ ID NO:102995 ' 3 ' frame 1 translation thing. In this translation thing, found the ORF of following coding: SEQ ID NO:10342.

The present invention includes the polypeptide that contains the amino acid sequence that is selected from SEQ ID NO:10338, SEQ NO:10339, SEQ NID NO:10340, SEQ ID NO:10341 and SEQ ID NO:10342. The present invention includes the polypeptide that has the sequence homogeny with the amino acid sequence that is selected from SEQ ID NO:10338, SEQ NO:10339, SEQ NID NO:10340, SEQ ID NO:10341 and SEQ ID NO:10342. The present invention includes the fragment of the polypeptide that contains the amino acid sequence that is selected from SEQ ID NO:10338, SEQ NO:10339, SEQ NID NO:10340, SEQ ID NO:10341 and SEQ ID NO:10342. In one embodiment, this fragment is not to be made of the amino acid sequence of known SARS virus or the amino acid sequence of known coronavirus fully.

In one embodiment, SEQ ID NOS:10338-10342 is used to fusion. Therefore, the initiation codon methionine is removed. The present invention includes amino acid sequence and be selected from SEQ ID NO:10343, SEQ ID NO:10344, SEQ ID NO:10345, SEQ ID NO:10346 and SEQ ID NO:10347.

In one embodiment, the present invention includes the amino acid sequence that is selected from SEQ ID NO:10338 and SEQ ID NO:10339. Below be the part BLAST result who GenBank is carried out SEQ ID NO:10338 retrieval:

The RNA polymerase (ORF1B) that instructs of gi|133593|sp|P18457|RRPB_CVPFS RNA

The RNA polymerase (Ec 2.7.7.48) that gi|93934|pir||A43489 RNA instructs-transmissible gastro-enteritis virus (fragment)

Gi|833161|emb|CAA37284.1| polymerase [TGE]

Length=533

Mark=131 (329) estimated value=3e-30

Homogeny=55/89 (61%), positive=69/89 (77%), space=1/89 (1%)

Inquiry: 1 MLWCKDGHVETFYPKLQASQAWQPGVAMPNLYKMQRMLLEKCDLQNYGENAVIPKG IMMN 60

            MLWC++ H++TFYP+LQ+++ W PG +MP LYK+QRM LE+C+L NYG    +P GI  N

Target: 217 MLWCENSHIKTFYPQLQSAE-WNPGYSMPTLYKIQRMCLERCNLYNYGAQVKLPDG ITTN 275

Inquiry: 61 VAKYTQLCQYLNTLTLAVPSNMRVIHFGA 89

            V KYTQLCQYLNT TL VP MRV+H GA

Target: 276 VVKYTQLCQYLNTTTLCVPHKMRVLHLGA 304

These results show that SEQ ID NO:10338 is similar on function to the RNA polymerase that transmissible gastro-enteritis virus RNA instructs.

The part BLAST result who GenBank is carried out SEQ ID NO:10339 retrieval is as follows:

Gb|AAL57305.1| replicase [bovine coronavirus]

Length=7094

Mark=139 (351) estimated value=7e-33

Homogeny=64/108 (59%), positive=78/108 (72%)

Inquiry: 1 MSVISKVVKVTIDYAEISFMLWCKDGHVETFYPKLQASQAWQPGVAMPNLYKMQRM LLEK 60

            M++SKVV V  +D+ +  FMLWC D  V TFYP+LQA+  W+PG +MP LYK    +E+

Target: 6760 LNCVSKVVNVNVDFKDFQFMLWCNDEKVMTFYPRLQAASDWKPGYSMPVLYKYLNS PMER 6819

Inquiry: 61 CDLQNYGENAVIPKGIMMNVAKYTQLCQYLNTLTLAVPSNMRVIHFGA 108

              L NYG+   +P G MMNVAKYTQLCQYLNT TLAVP NMRV+H GA

Target: 6820 VSLWNYGKPVTLPTGCMMNVAKYTQLCQYLNTTTLAVPVNMRVLHLGA 6867

These results show that SEQ ID NO:10339 is similar on function to the replicase of bovine coronavirus.

SARS virus has polymorphism at the Glu-20 residue of SEQ ID NO:10338. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:10338, wherein said polypeptide comprises the amino acid sequence that is selected from ASQAW (SEQ ID NO:10348) and ASRAW (SEQ ID NO:10349). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:10338, wherein said fragment comprises the amino acid sequence that is selected from SEQ ID NO:10348 and SEQ ID NO:10349.

SARS virus has polymorphism at the Ser-80 residue of SEQ ID NO:10338. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:10338, wherein said polypeptide comprises the amino acid sequence that is selected from VPSNM (SEQ ID NO:10350) and VPTNM (SEQ ID NO:10351). The present invention includes the fragment of the polypeptide that contains SEQID NO:10338, wherein said fragment comprises the amino acid sequence that is selected from SEQ ID NO:10350 and SEQ ID NO:10351.

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the polynucleotide sequence of probe of the method for SARS virus. The present invention includes the polynucleotide sequence that contains one or more primer sequences that table 32 identifies. The present invention also comprises the polynucleotide sequence of the complementary series that contains one or more primer sequences that table 32 identifies.

The invention provides SARS polynucleotide sequence SEQ ID NO:10505. Figure 127 has shown all 6 reading frames (also can referring to Figure 132) of this sequence. The composition amino acid sequence of Figure 127 contains at least 4 amino acid, and they are used as SEQ ID NOS:10506-10570 and list.

The present invention includes the polynucleotide sequence that contains SEQ ID NO:10505. The present invention also provides the polynucleotide sequence that has the sequence homogeny with SEQ ID NO:10505. The present invention also provides the polynucleotide sequence that contains SEQ ID NO:10505 fragment. In one embodiment, this polynucleotide passage is not to be made of known SARS virus polynucleotide sequence or known coronavirus polynucleotide sequence fully.

The present invention includes the amino acid sequence by the polynucleotide sequence coding of SEQ ID NO:10505, comprise the amino acid sequence shown in Figure 127, especially those are selected from the amino acid sequence of SEQ ID NOS:10506-10570. Preferred described amino acid sequence comprises SEQ ID NO:10532 and/or SEQ ID NO:10533.

The present invention also provides the amino acid sequence that has the sequence homogeny with the amino acid sequence by SEQ ID NO:10505 coding. The invention provides and the amino acid sequence that is selected from sequence shown in Figure 127, especially SEQ ID NOS:10506-10570 has the amino acid sequence of sequence homogeny.

The present invention also provides the fragment by the coded amino acid sequence of SEQ ID NO:10505. The present invention also provides the fragment of the amino acid sequence that is selected from SEQ ID NOS:10506-10570. In one embodiment, this fragment is not to be made of the known amino acid sequence of SARS virus or the known amino acid sequence of coronavirus fully.

In one embodiment, the present invention includes the polypeptide that contains from the amino acid sequence of 5 ' 3 ' frames 3 of Figure 127. The ORF of some codings is in this translation thing: SEQ ID NO:10533; SEQ ID NO:10571; SEQ ID NO:10572; SEQ ID NO:10573; SEQ ID NO:10574.

The present invention includes the peptide sequence that contains the amino acid sequence that is selected from SEQ ID NO:10533, SEQ ID NO:10571, SEQ ID NO:10572, SEQ ID NO:10573 and SEQ ID NO:10574. The present invention includes the polypeptide that has the sequence homogeny with the amino acid sequence that is selected from SEQ ID NO:10533, SEQ ID NO:10571, SEQ ID NO:10572, SEQ ID NO:10573 and SEQ ID NO:10574. The present invention includes the fragment of the peptide sequence that contains the amino acid sequence that is selected from SEQ ID NO:10533, SEQ ID NO:10571, SEQ ID NO:10572, SEQ ID NO:10573 and SEQ ID NO:10574.

The part BLAST result who GenBank is carried out SEQ ID NO:10533 retrieval is as follows:

Gi|7739601|gb| is from F68926.1|AF207902_11 nucleocapsid protein [murine hepatitis virus ML-11 strain]

Length=451

Mark=147 (370) estimated value=3e-34

Homogeny=102/252 (40%), positive=137/252 (54%), space=18/252 (7%)

Inquiry: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKEL SPRW 106

            SWF+ +TQ  K +E +F +GQGVPI +     +Q GY +R  RR  +  DG +K+L PRW

Target: 63 SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQL LPRW 122

Inquiry: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQG TTLP 166

            YFYYLGTGP A   YG +EG +VWVA++ A       +  R+P+++ A   +   GT LP

Target: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTRFAPG TVLP 182

Inquiry: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLL DRLN 226

            +GFY EGS   + AS   S     N       SS    PA          +A L+L +L

Target: 183 QGFYVEGSGRSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVL AKLG 242

Inquiry: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPE QTQG 282

            +        GQ+Q    VTK+SA E  +    KPRQKRT  KQ  V Q FG+RGP Q

Target: 243 K------DAGQPKQ---VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPN Q---290

Inquiry: 283 NFGDQDLIRQGT 294

            NFG  ++++ GT

Target: 291NFGGSEMLKLGT 302

Gi | 3132999|gb|AAc16422.1| nucleocapsid protein [murine hepatitis virus strain 2]

Length=451

Mark=147 (370) estimated value=3e-34

Homogeny=102/252 (40%), positive=137/252 (54%), space=18/252 (7%)

Inquiry: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKEL SPRW 106

            SWF+ +TQ  K +E +F +GQGVPI +     +Q GY+ R  RR  +  DG+K+L PRW

Target: 63 SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQL LPRW 122

Inquiry: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQG TTLP 166

            YFYYLGTGP A   YG + EG+VWVA++ A       +  R+P+++ A   +   GT LP

Target: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTKFAPG TVLP 182

Inquiry: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLL DRLN 226

            +GFY EGS   + AS    S    N       SS    PA          +A L+L +L

Target: 183 QGFYVEGSGKSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVL AKLG 242

Inquiry: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPE QTQG 282

            +        GQ +Q   VTK+SA E +     KPRQKRT  KQ  V Q FG+RGP Q

Target: 243 K------DAGQPKQ---VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPN Q---290

Inquiry: 283 NFGDQDLIRQGT 294

            NFG  ++++GT

Target: 291 NFGGSEMLKLGT 302

The gi|127877|sp|P03417|NcAP_CVMJH nucleocapsid protein

Gi|74859|pir||VHIHMJ nucleocapsid protein-murine hepatitis virus (strain JHM)

Gi|58973|emb|CAA25497.1| nucleocapsid protein [murine hepatitis virus]

Length=455

Mark=146 (369) estimated value=4e-34

Homogeny=110/254 (43%), positive=142/254 (55%), space=22/254 (8%)

Inquiry: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKEL SPRW 106

            SWF+ +TQ  K +E +F +GQGVPI        Q GY+ R  RR  +  DG+ K+L PRW

Target: 67 SWFSGITQFQKGKEFQFAQGQGVPIANGIPASQQKGYWYRHNRRSFKTPDGQQKQL LPRW 126

Inquiry: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQG TTLP 166

            YFYYLGTGP A   YG + EG+VWVA++ A       I  R+P+++ A   +   GT LP

Target: 127 YFYYLGTGPYAGAEYGDDIEGVVWVASQQAETRTSADIVERDPSSHEAIPTRFAPG TVLP 186

Inquiry: 167 KGFYAEGSRGGSQASSRSSSR--SRGNSRNSTPGSSRGNSPARMASGGGETALALL LLDR 224

            +GFY EGS G S  +SRS SR  SRG   N    SS    PA          +A L+L +

Target: 187 QGFYVEGS-GRSAPASRSGSRPQSRG-PNNRARSSSNQRQPASTVKPDMAEEIAAL VLAK 244

Inquiry: 225 LNQLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRG PEQT 280

            L+         GQ +Q  VTK+SA E   +    KPRQKRT  KQ  V Q FG+RGP Q

Target: 245 LGK------DAGQPKQ---VTKQSAKEVRQKILNKPRQKRTPNKQCPVQQCFGKRG PNQ-294

Inquiry: 281 QGNFGDQDLIRQGT 294

              NFG  ++++ GT

Target: 295--NFGGPEMLKLGT 306

Gi|6625766|gb|AAF19389.1|AF201929_7 nucleocapsid protein [murine hepatitis virus strain 2]

Gi|7769348|gb|AAF69338.1|AF208066_11 nucleocapsid protein [murine hepatitis virus]

Length=451

Mark=146 (368) estimated value=5e-34

Homogeny=102/252 (40%), positive=137/252 (54%), space=18/252 (7%)

Inquiry: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKEL SPRW 106

            SWF+ +TQ  K +E +F +GQGVPI +     +Q GY+ R  RR  +  DG+ K+L PRW

Target: 63 SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQL LPRW 122

Inquiry: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQG TTLP 166

            YFYYLGTGP A   YG + EG+VWVA++ A       +  R+P+++ A   +   GT LP

Target: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTRFAPG TVLP 182

Inquiry: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLL DRLN 226

            +GFY EGS   + AS   S        N    SS    PA          +A L+L +L

Target: 183 QGFYVEGSGRSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVL AKLG 242

Inquiry: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPE QTQG 282

            +        GQ +Q   VTK+SA E  +    KPRQKRT  KQ  V Q FG+RGP Q

Target: 243 K------DAGQPKQ---VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPN Q---290

Inquiry: 283 NFGDQDLIRQGT 294

            NFG  ++++ GT

Target: 291 NFGGSEMLKLGT 302

The Nucleocapsid protein [pigs haemagglutinating encephalomyelitis virus] of gi|21734854|gb|AAM77005.1|AF481863_7 phosphorylation

Length=449

Mark=145 (366) estimated value=8e-34

Homogeny=107/253 (42%), positive=145/253 (57%), space=18/253 (7%)

Inquiry: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKEL SPRW 106

            SWF+ +TQ  K +E  F  GQGVPI       +  GY+ R  RR  +  DG  ++L PRW

Target: 64 SWFSGITQFQKGKEFEFAEGQGVPIAPGVPATEAKGYWYRHNRRSFKTADGNQRQL LPRW 123

Inquiry: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGA-LNTPKDHIGTRNPNNNAATVLQLPQ GTTL 165

            YFYYLGTGP A   YG + +G+ WVA+  A +NTP D I  R+P+++ A   + P GT L

Target: 124 YFYYLGTGPHAKHQYGTDIDGVFWVASNQADINTPAD-IVDRDPSSDEAIPTRFPP GTVL 182

Inquiry: 166 PKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLL LDRL 225

            P+G+Y EGS G S  +SRS+SR+  N   S    SR NS  R ++ G    +A    D++

Target: 183 PQGYYIEGS-GRSAPNSRSTSRA-PNRAPSAGSRSRANSGNRTSTPGVTPDMA----DQI 236

Inquiry: 226 NQLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGP EQTQ 281

               L    GK    + Q VTK++A E  +    KPRQKR+  KQ  V Q FG+RGP Q

Target: 237 ASLVLAKLGK-DATKPQQVTKQTAKEVRQKILNKPRQKRSPNKQCTVQQCFGKRGP NQ--293

Inquiry: 282 GNFGDQDLIRQGT 294

             NFG  ++++GT

Target: 294-NFGGGEMLKLGT 305

Gi|23295765|gb|AAL80036.1| nucleocapsid protein [pigs haemagglutinating encephalomyelitis virus]

Length=449

Mark=145 (365) estimated value=1e-33

Homogeny=107/253 (42%), positive=145/253 (57%), space=18/253 (7%)

Inquiry: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKEL SPRW 106

            SWF+ +TQ  K +E  F  GQGVPI       +  GY+ R  RR  +  DG  ++L PRW

Target: 64 SWFSGITQFQKGKEFEFAEGQGVPIAPGVPSTEAKGYWYRHNRRSFKTADGNQRQL LPRW 123

Inquiry: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGA-LNTPKDHIGTRNPNNNAATVLQLPQ GTTL 165

            YFYYLGTGP A   YG + +G +WVA + A +NTP D I  R+P+++ A   + P GT L

Target: 124 YFYYLGTGPHAKDQYGTDIDGVFWVASNQADINTPAD-IVDRDPSSDEAIPTRFPP GTVL 182

Inquiry: 166 PKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLL LDRL 225

            P+G+Y EGS G S  +SRS+SR+  N   S    SR NS R++G    +A  D++

Target: 183 PQGYYIEGS-GRSAPNSRSTSRA-PNRAPSAGSRSRANSGNRTSTPGVTPDMA----DQI 236

Inquiry: 226 NQLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGP EQTQ 281

              L     GK    + Q VTK++A E  +    KPRQKR+  KQ  V Q FG+RGP Q

Target: 237 ASLVLAKLGK-DATKPQQVTKQTAKEVRQKILNKPRQKRSPNKQCTVQQCFGKRGP NQ--293

Inquiry: 282 GNFGDQDLIRQGT 294

            NFG   ++++GT

Target: 294-NFGGGEMLKLGT 305

These results show on SEQ ID NO:10533 and the coronavirus nucleocapsid protein function similar.

In one embodiment, the present invention includes the amino acid sequence of 5 ' 3 ' frames 3 of Figure 127, for example SEQ ID NOs:10506-10514. The ORF of some codings is SEQ ID NO:10575-10578 in this zone.

Therefore, the present invention includes the polypeptide that contains the amino acid sequence that is selected from SEQ ID NO:10575, SEQ ID NO:10576, SEQ ID NO:10577 and SEQ ID NO:10578. The present invention includes and the polypeptide that is selected from SEQ ID NO:10575, SEQ ID NO:10576, SEQ ID NO:10577 and SEQ ID NO:10578 sequence and has the amino acid sequence of sequence homogeny. The present invention includes the fragment of the peptide sequence that contains the amino acid sequence that is selected from SEQ ID NO:10575, SEQ ID NO:10576, SEQ ID NO:10577 and SEQ ID NO:10578.

In one embodiment, the present invention includes the amino acid sequence of 3 ' 5 ' frames 2 that contain Figure 127, for example the polypeptide of SEQ ID NOs:10547-10559. ORF in this zone is SEQ ID NO:10579.

The present invention includes the polypeptide that contains SEQ ID NO:10579 amino acid sequence. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:10579. The present invention includes the fragment of the polypeptide of the amino acid sequence that contains SEQ ID NO:10579.

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the polynucleotide sequence of probe of the method for SARS virus. The present invention includes the polynucleotide sequence that contains one or more primer sequences that table 33 identifies. The present invention also comprises the polynucleotide sequence of the complementary series that contains one or more primer sequences that table 33 identifies.

The present invention includes the polynucleotide sequence that contains SEQ ID NO:11323. A polypeptide by SEQ ID NO:11323 coding is SEQ ID NO:11324.

The present invention includes the polypeptide that contains SEQ ID NO:11324, has the sequence of sequence homogeny with the fragment of SEQ ID NO:11324 and SEQ ID NO:11324. The present invention includes the fragment of SEQ ID NO:11324, wherein said polypeptide fragment starts from methionine.

Therefore, the present invention includes the polynucleotide sequence that contains SEQ ID NO:11323. The present invention also provides the polynucleotide sequence that has the sequence homogeny with SEQ ID NO:11323. The present invention also provides the polynucleotide sequence that contains SEQ ID NO:11323 fragment. In one embodiment, this polynucleotide passage is not to be made of known SARS virus polynucleotide sequence or known coronavirus polynucleotide sequence fully.

The present invention includes the amino acid sequence by polynucleotide sequence SEQ ID NO:11323 coding, comprise the amino acid sequence of SEQ ID NO:11324.

The present invention also provides the amino acid sequence that has the sequence homogeny with the amino acid sequence of SEQ ID NO:11323 coding. The invention provides the amino acid sequence that has the sequence homogeny with SEQ ID NO:11324.

The invention provides the fragment by the amino acid sequence of SEQ ID NO:11323 coding. The present invention also provides the fragment of the amino acid sequence of SEQ ID NO:11324. In one embodiment, this fragment is not that complete amino acid sequence by the amino acid sequence that consists of known SARS virus or known coronavirus consists of.

The present invention also comprise can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the polynucleotide sequence of probe of the method for SARS virus. The present invention includes the polynucleotide sequence that contains the one or more primer sequences that are accredited as SEQ ID NO:11325-11440 (left-hand component) and SEQ ID NOS:11441-11551 (right-hand component). The present invention also comprises the polynucleotide sequence that contains the one or more primer sequence complementary series that are accredited as SEQ ID NO:11325-11551.

The present invention includes the polypeptide that contains SEQ ID NO:11552. SARS virus has polymorphism at isoleucine residue Ile-324. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11552, wherein said polypeptide comprises and is selected from lower group amino acid sequence: YSYAI (SEQ ID NO:11553), SYAIH (SEQ ID NO:11554), YAIHH (SEQ ID NO:11555), IHHDK (SEQ ID NO:11556), SYAI (SEQ ID NO:11557), YAIH (SEQ ID NO:11558), AIHH (SEQ ID NO:11559), IHHD (SEQ ID NO:11560), YAI, AIH and IHH. The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11552, wherein said fragment comprises and is selected from lower group amino acid sequence: YSYAI (SEQ ID NO:11553), SYAIH (SEQ ID NO:11554), YAIHH (SEQ ID NO:11555), IHHDK (SEQ ID NO:11556), SYAI (SEQ ID NO:11557), YAIH (SEQ ID NO:11558), AIHH (SEQ ID NO:11559), IHHD (SEQ ID NO:11560), YAI, AIH and IHH.

The present invention includes the polypeptide that contains the amino acid sequence that is selected from SEQ ID NO:11561 and SEQ ID NO:11562. The present invention includes the fragment of the polypeptide of the amino acid sequence that is selected from SEQ ID NO:11561 and SEQ ID NO:11562.

The present invention includes diagnostic kit, the polypeptide of the amino acid sequence that contains at least a SEQ of being selected from ID NOS:11561 and 11562 is housed in the box. The present invention includes the diagnostic kit of the polynucleotide sequence that contains coded polypeptide, wherein said polypeptide contains at least one amino acid sequence that is selected from SEQ ID NO:11561-11562. The present invention includes a kind of immunogenic composition, wherein comprise the polypeptide of the amino acid sequence that contains at least a SEQ of being selected from ID NOS:11561 and 11562. The present invention includes the antibody of the polypeptide that can identify the amino acid sequence that contains at least a SEQ of being selected from ID NOS:11561 and 11562.

The present invention includes polynucleotide sequence SEQ ID NO:11563 or its fragment or have the sequence of sequence homogeny with it. Can be shown in Figure 128 from the peptide sequence of SEQ ID NO:11563 translation. In the composition amino acid sequence of Figure 128 at least 4 amino acid are arranged, they are used as SEQ ID NOS:11564-11617 and list.

The present invention includes the peptide sequence of the sequence that is selected from Figure 128, or its fragment or have the sequence of sequence homogeny with it, for example SEQ ID NOS:11563-11617.

Peptide sequence among the SEQ ID NO:11600 is SEQ ID NO:11618. The present invention includes the polypeptide that contains SEQ ID NO:11618 or its fragment or have the sequence of sequence homogeny with it.

Peptide sequence among the SEQ ID NO:11602 is SEQ ID NO:11641. The present invention includes the polypeptide that contains SEQ ID NO:11641 or its fragment or have the sequence of sequence homogeny with it.

Peptide sequence among the SEQ ID NO:11609 is SEQ ID NO:11619.

The present invention includes the polynucleotides of the following sequence of coding: (i) be selected from lower group amino acid sequence: the amino acid sequence of (1) Figure 128, especially SEQ ID NOS:11564-11617; (2) SEQ ID NO:11618; (3) SEQ ID NO:11619, or (ii) its fragment. The present invention includes the diagnostic kit that contains one or more these protein. The present invention includes the diagnostic kit of the polynucleotide sequence that contains one or more these peptide sequences of encoding. The present invention includes the antibody that to identify one or more peptide sequences.

SARS virus can have polymorphism on the isoleucine residue Ile-326 of SEQ ID NO:11620 (Chi-PEP3). The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11620, wherein said polypeptide comprises and is selected from YAIHH (SEQ ID NO:11621) and YATThe amino acid sequence of HH (SEQ ID NO:11622). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11620, wherein said fragment comprises and is selected from YAIHH (SEQ ID NO:11621) and YATThe amino acid sequence of HH (SEQ ID NO:11622).

SARS virus can have polymorphism on the glutamine residue Gln-830 of SEQ ID NO:11620. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11620, wherein said polypeptide comprises and is selected from ASQAW (SEQ ID NO:11623) and ASRThe amino acid sequence of AW (SEQ ID NO:11624). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11620, wherein said fragment comprises and is selected from ASQAW (SEQ ID NO:11623) and ASRThe amino acid sequence of AW (SEQ ID NO:11624).

SARS virus can have polymorphism on the asparagicacid residue Asp-935 of SEQ ID NO:11620. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11620, wherein said polypeptide comprises and is selected from DADST (SEQ ID NO:11625) and DAYThe amino acid sequence of ST (SEQ ID NO:11626). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11620, wherein said fragment comprises and is selected from DADST (SEQ ID NO:11625) and DAYThe amino acid sequence of ST (SEQ ID NO:11626).

SARS virus can have polymorphism on the serine residue Ser-577 of SEQ ID NO:11627 (Chi-PEP4). The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11627, wherein said polypeptide comprises and is selected from PCSFG (SEQ m NO:11628) and PCAThe amino acid sequence of FG (SEQ ID NO:11629). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11627, wherein said fragment comprises and is selected from PCSFG (SEQ ID NO:11628) and PCAThe amino acid sequence of FG (SEQ ID NO:11629).

SARS virus can have polymorphism on the valine residue Val-68 of SEQ ID NO:11630 (Chi-PEP8). The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11630, wherein said polypeptide comprises and is selected from LAVVY (SEQ ID NO:11631) and LAAThe amino acid sequence of VY (SEQ ID NO:11632). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11630, wherein said fragment comprises and is selected from LAVVY (SEQ ID NO:11631) and LAAThe amino acid sequence of VY (SEQ ID NO:11632).

SARS virus can have polymorphism on the isoleucine residue Ile-50 of SEQ ID NO:11633 (Chi-PEP13). The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11633, wherein said polypeptide comprises and is selected from NNIAS (SEQ ID NO:11634) and NNTThe amino acid sequence of AS (SEQ ID NO:11635). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11633, wherein said fragment comprises and is selected from NNIAS (SEQ ID NO:11634) and NNTThe amino acid sequence of AS (SEQ ID NO:11635).

SARS virus can have polymorphism on the serine residue Ser-943 of SEQ ID NO:11636. The present invention includes and contain the polypeptide that has the amino acid sequence of sequence homogeny with SEQ ID NO:11636, wherein said polypeptide comprises and is selected from AVSAC (SEQ ID NO:11637) and AVGThe amino acid sequence of AC (SEQ ID NO:11638). The present invention includes the fragment of the polypeptide that contains SEQ ID NO:11636, wherein said fragment comprises and is selected from AVSAC (SEQ ID NO:11637) and AVGThe amino acid sequence of AC (SEQ ID NO:11638).

The present invention includes polynucleotides SEQ ID NO:11639, or its fragment or have the sequence of sequence homogeny with it. The present invention includes the polypeptide by the listed polynucleotide sequence coding of SEQ ID NO:11639, or its fragment or have the peptide sequence of sequence homogeny with it.

The present invention includes the listed polynucleotides of SEQ ID NO:11640, or its fragment or have the sequence of sequence homogeny with it. The present invention includes the polypeptide by the listed polynucleotide sequence coding of SEQ ID NO:11640, or its fragment or have the peptide sequence of sequence homogeny with it.

The present invention includes each polynucleotides of identifying above. The present invention includes each listed in sequence table polynucleotides. The present invention also comprises the polynucleotides that have the sequence homogeny with each polynucleotides of identifying above. Sequence homogeny degree is preferably greater than 50% (for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher).

The present invention includes the polynucleotide sequence that contains each polynucleotide sequence fragment of identifying above. Described fragment should contain specific SEQ ID NO: n continuity polynucleotides at least, according to sequence, n can be 7 or more (for example, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,45,50,55,60,65,70,80,90,100,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300 or more).

The present invention includes each amino acid sequence by each polynucleotide sequence of coding of identifying above. The present invention includes each amino acid sequence by listed each polynucleotide sequence in the sequence table. The present invention also comprises the amino acid sequence that has the sequence homogeny with the amino acid sequence by each polynucleotide sequence coding of identifying above. Sequence homogeny degree is preferably greater than 50% (for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher). The present invention also comprises the fragment by the amino acid sequence of each polynucleotide sequence coding of identifying above. Described fragment should contain specific SEQ ID NO: n continuous amino acid at least, according to sequence, n can be 7 or more (for example, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,45,50,55,60,65,70,80,90,100,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300 or more).

The present invention includes each amino acid sequence of identifying above. The present invention includes each listed in sequence table amino acid sequence. The present invention also comprises the amino acid sequence that has the sequence homogeny with each amino acid sequence of identifying above. Sequence homogeny degree is preferably greater than 50% (for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher).

The present invention also comprises the fragment of the amino acid sequence of identifying above. Described fragment should contain specific SEQ ID NO: n continuous amino acid at least, according to sequence, n can be 7 or more (for example, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,45,50,55,60,65,70,80,90,100,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300 or more).

The present invention includes each amino acid sequences encoded polynucleotides that coding is identified above. The present invention includes each listed amino acid sequences encoded polynucleotides in the code sequence tabulation. The present invention comprises that also the polynucleotides of each amino acid sequence of identifying above with coding have the polynucleotides of sequence homogeny. Sequence homogeny degree is preferably greater than 50% (for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher).

The present invention also comprises the fragment of the polynucleotides of each amino acid sequence that coding is identified above. Described fragment should contain specific SEQ ID NO: n continuous polynucleotides at least, according to sequence, n can be 7 or more (for example, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,45,50,55,60,65,70,80,90,100,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300 or more).

As described in greater detail below, can contain at least 4 or 8 contiguous nucleotides from polynucleotide sequence of the present invention as the polynucleotides of primer and/or probe, at least 14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 contiguous nucleotides for example, but and about 50,75,100,200 contiguous nucleotides or more of as many as. Because 6-8 nucleotides is the length that can work, the sequence of 10-12 nucleotides is preferably, about 13,14,15,16,17,18,19,20 21 or more nucleotide pair be best in hybridization.

In one embodiment, the present invention relates to not is complete polynucleotides or amino acid sequence by known SARS virus, or polynucleotides and the amino acid sequence of known coronavirus polynucleotides or amino acid sequence formation. In one embodiment, polynucleotides of the present invention and amino acid sequence are not to be made of sequence SEQ ID NO:1 fully. In another embodiment, polynucleotides of the present invention and amino acid sequence are not to be made of sequence SEQ ID NO:2 fully. SEQ ID NO:9967 is the SARS genome sequence (GenBank:AY310120) of Frankfurt (FRA) separator. Compare with SEQ ID NO:1, it is different on nucleotides 2546,2590,11437,18954,19073,20585,20899,23209,24922,26589 and 28257; Compare with SEQ ID NO:2, it is different on nucleotides 2560,7922,11451,16625,18968 and 19067. Since the initial submission of the application, other genome sequence can following accession number obtain from GenBank: AY559097, AY559096, AY559095, AY559094, AY559093, AY559092, AY559091, AY559090, AY559089, AY559088, AY559087, AY559086, AY559085, AY559084, AY559083, AY559082, AY559081, AY274119, AY323977, AY291315, AY502932, AY502931, AY502930, AY502929, AY502928, AY502927, AY502926, AY502925, AY502924, AY502923, AY291451, AY390556, AY395003, AY395002, AY395001, AY395000, AY394999, AY394998, AY394997, AY394996, AY394995, AY394994, AY394993, AY394992, AY394991, AY394990, AY394989, AY394987, AY394986, AY394985, AY394983, AY394979, AY394978, AY508724, AY394850, AY463059, AY463060, AY313906, AY310120, AY461660, AY485278, AY485277, AY345988, AY345987, AY345986, AY282752, AY357076, AY357075, AY350750, AY304495, AY304488, AY304486, AY427439, AY283798, AY278491, AY278489, AY362699, AY362698, AY283797, AY283796, AY283795, AY283794, AY278741, AY351680, AP006561, AP006560, AP006559, AP006558, AP006557, AY278554, AY348314, AY338175, AY338174, AY321118, AY279354, AY278490, AY278487, AY297028, AY278488 and NC_004718.

In another embodiment, the present invention relates to encode not polynucleotides with the protein generation immunological cross-reaction protein of MHV, bovine coronavirus or avian infectious bronchitis virus. In another embodiment, the present invention relates to not protein with the protein generation immunological cross-reaction of MHV, bovine coronavirus or avian infectious bronchitis virus.

Each polynucleotides that the above identifies can be used for the coded portion fusion. Therefore, the present invention includes one or more polynucleotides of identifying above, wherein, the polynucleotides of coding initiation codon are removed. The present invention also comprises one or more amino acid of identifying above, and wherein, initial methionine is removed.

Above-mentioned any polynucleotides or amino acid sequence can be used for treating or preventing the vaccine of SARS virus infection, comprise as SARS virus antigen. In addition, above-mentioned any polynucleotides or amino acid sequence can be used for diagnosticum, kit (comprising this reagent) and be used for diagnosing or the identification of organism sample in whether have the method for SARS virus.

SARS virus antigen of the present invention comprise with following protein in one or more have the polypeptide of 99%, 95%, 90%, 85% or 80% homology: non-structural protein 2 (NS2); Hemagglutinin-esterase glycoprotein (HE) (being also referred to as E3), spike glycoprotein (S) (being also referred to as E2), non-structural area 4 (NS4), coating (membranelle) albumen (E) (being also referred to as sM), membrane glycoprotein (M) (being also referred to as E1), nucleocapsid phosphoprotein (N) or RNA-directed RNA polymerase (pol).

To the biological visible Fields Virology (second edition) that is described in detail of coronavirus, Fields etc. (volume), B.N.Raven Press, New York, NY., the 35th chapter.

Listed other example of SARS virus separator in the following examples 1. The present invention includes each polypeptide and the polynucleotide sequence identified among the embodiment 1. In addition, the present invention includes and contain among the embodiment 1 one or more polypeptide of identifying or the bacterin preparation of polynucleotide sequence. The present invention includes diagnosticum, the kit (comprising this reagent) that uses the polypeptide identified among one or more embodiment 1 or polynucleotide sequence and be used for diagnosing or the identification of organism sample in whether have the method for SARS virus. The present invention includes with the combination of little molecule viral inhibitors and little molecule viral inhibitors and kit and treat or prevent SARS virus to infect to treat the method for SARS. Polypeptide or polynucleotides that described micromolecular inhibitor is identified in can selectively targeted one or more embodiment 1.

The below is the further narration to term used herein.

" Respirovirus " refers to infect the virus of human airway here. Be applicable to Respirovirus antigen of the present invention and comprise severe acute respiratory syndrome virus, coronavirus, influenza virus, ERC group virus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus(RSV) (RSV), adenovirus, super Pneumovirinae and rhinovirus.

Term " polypeptide ", " protein " and " amino acid sequence " typically refer to the amino acid residue polymer here, do not have the minimum product length restriction. Therefore, peptide, oligopeptides, dimer, polymer etc. are included in the definition. Full-length proteins and its fragment are included in the definition. Can have at least 3,4,5,6,7,8,9,10,11,12,13,14 or even 15 amino acid to the useful minimum polypeptide fragment of the present invention. Usually, to the useful polypeptide of the present invention the maximum length that is fit to required application can be arranged. Usually, described maximum length does not have strict standard, and is that the technical staff who is proficient in this field is not difficult to select.

Polypeptide of the present invention can prepare by the whole bag of tricks, for example by chemical synthesis (at least part of), by with the longer polypeptide of protease digestion, by the translation of RNA, by from cell culture (such as recombinant expressed), from organism self (such as from viral cultures or directly from the patient), from purifying such as clone sources. Factory length comprises external chemical synthesis less than the method for optimizing of 40 amino acid whose peptides, and (Bodanszky (1993) " peptide composition principle " (Principles of Peptide Synthesis) (ISBN:0387564314); Fields etc. (1997) Enzymology method 289: solid-phase peptide is synthesized .ISBN:0121821900). The solid phase method of peptide synthesis particularly preferably is such as the chemical method based on t-Boc or Fmoc (Chan ﹠ White (2000) " the Fmoc solid-phase peptide is synthetic " (Fmoc Solid Phase Peptide Synthesis) ISBN:0199637245). Also can partly or entirely use the enzymatic synthesis method (Kullmann (1987) " utilizing the enzymatic synthesis peptide " (Enzymatic Peptide Synthesis), ISBN:0849368413). Except chemical synthesis, also can use biological synthesis process, for example can make polypeptide by translation. This can carry out in external or body. Biological method only limits to make based on the amino acid whose polypeptide of L-usually; but available multiple translating mechanism (for example aminoacyl tRNA molecule) is introduced D-amino acid (or other alpha-non-natural amino acid, such as iodotyrosine or methylphenylalanine, nitrine high lactamine etc.) (Ibba (1996) Biotechizol Geizet Eng Rev 13:197-216). When comprising D-amino acid, preferably use chemical synthesis. The C-end of polypeptide of the present invention and/or N-end can by covalent modification, especially when they are used for using in the body, for example can look like Fuzeon^TMProduct is modified by adding acetyl group or carbamyl like that.

The derivative that when mentioning polypeptide, also comprises amino acid sequence of the present invention. This derivative is modified such as glycosylation, acetylation, phosphorylation etc. after can comprising the expression of polypeptide. Amino acid derivativges also can comprise the modification to native sequences, such as disappearance, insert and replace (conservative in nature), as long as albumen is kept required activity. These modifications can be artificial, as by rite-directed mutagenesis, maybe can be accidental, such as the mistake that the host suddenlys change or pcr amplification causes by generating albumen. In addition, modification can cause one or more following effects: reduce toxicity; Be easy to cell processing (for example, secretion, antigen presentation etc.); And be easy to present B-cell and/or T-cell.

" fragment " or " protein " only refers to the polypeptide that the part by the complete full-length polypeptide sequence of natural discovery and structure forms here. For example, fragment can comprise C-terminal deletion and/or the N-terminal deletion of certain protein.

" restructuring " albumen is the protein of making by recombinant DNA technology described here. Usually, then interested gene is cloned and expresses in the organism that transforms, as described below. Host living beings under expression condition expression alien gene to produce protein.

Term " polynucleotides " typically refers to a kind of nucleic acid molecules in the art. " polynucleotides " can comprise two strands and single stranded sequence, and it includes but not limited to cDNA, prokaryotes or the Eukaryotic mRNA of virus, geneome RNA and dna sequence dna (for example RNA virus and dna virus and retrovirus) or procaryotic DNA and the especially synthetic dna sequence dna of virus. This term also comprises and contains any known DNA and the sequence of RNA base analogue, and comprise modification to native sequences, such as deletion, adding and replacement (normally conservative in the natural situation), therapeutic or antigen protein as long as this nucleic acid molecules can be encoded. These modifications can be artificial, as by direct mutagenesis, maybe can be accidental, such as the sudden change of the host by generating antigen. Modification to polynucleotides can produce multiple effect, comprises and for example utilizes polypeptide product to express in host cell.

Polynucleotides of the present invention can prepare by several different methods, for example can be from genomic library or wholly or in part chemical synthesis of cDNA library (for example phosphoramidite synthetic method of DNA), with the long nucleic acid of nuclease (for example Restriction Enzyme) digestion, nucleic acid or the nucleotides that will lack couples together (such as with ligase or polymerase) etc.

Polynucleotides codified biologically active (for example, immunogenicity or therapeutic) protein or polypeptide. According to the character by the polypeptide of polynucleotide encoding, polynucleotides can comprise few to 10 nucleotides, for example polynucleotides of coding for antigens.

When being used for polynucleotides or polypeptide, " separate " refers to that mentioned molecule is to separate and dissociate to obtain from the natural complete organism that contains this molecule, perhaps, when these polynucleotides or polypeptide when not being natural, the finger that separates is substantially free of other large biological molecule, thereby described polynucleotides or polypeptide just can be used for required purpose. Polynucleotides of the present invention are preferably the polynucleotides that separate and the polypeptide that separates with polypeptide.

As known in the art, " antibody " comprises that one or more pass through the biological part that chemistry or physical method can be combined with epi-position of polypeptide of interest. Antibody of the present invention comprises the antibody of specific binding SARS virus antigen. Term " antibody " comprises the antibody available from polyclone and monoclonal goods, and: hybridization (chimeric) antibody molecule (referring to for example, Winter etc. (1991) Nature 349:293-299; With U.S. Patent No. 4816567; F (ab ')₂And F (ab) fragment; F_vMolecule (non-covalent heterodimer, referring to for example, Inbar etc. (1972) Proc Natl Acad Sci USA 69:2659-2662; With (1980) Biochem 19:4091-4096 such as Ehrlich); ScFv molecule (sFv) (referring to for example, Huston etc. (1988) Proc Natl Acad Sci USA 85:5897-5883); Dimerization and trimerization antibody fragment construction; Corpusculum (minibody) (referring to for example, Pack etc. (1992) Biochem 31:1579-1584; Cumber etc. (1992) J Immunology 149B:120-126); Humanized antibody molecules (referring to for example, Riechmann etc. (1988) Nature 332:323-327; Verhoeyan etc. (1988) Science 239:1534-1536; With disclosed UK Patent Application GB 2276169 on September 21st, 1994); And any function fragment of these molecules, wherein, this fragment has kept the immune binding characteristic of parental generation antibody molecule. Term " antibody " also comprises by nconventional method, for example the antibody that obtains of phage display.

Term " monoclonal antibody " refers to comprise the antibody compositions of homology antibody population here. This term is not subjected to the restriction in antibody type or source, nor limited by the manufacture method of antibody. Therefore, this term comprises the antibody available from the muroid hybridoma, also comprises the human monoclonal antibodies that employment rather than murine hybridoma obtain. Referring to for example, Cote etc., " monoclonal antibody and treatment of cancer " (Monoclonal Antibodies and Cancer Therapy), Alan R.Liss, 1985, p77.

" immunogenic composition " refers to contain the composition of antigenicity molecule here, this composition is used for the experimenter will causes the experimenter that antigen molecule interested is produced body fluid and/or cellullar immunologic response reaction. Immunogenic composition can be introduced directly into the experimenter, for example by in injection, suction, oral, the nose or other non-enteron aisle drug delivery route, mucous membrane or transdermal (for example in the rectum or in the vagina) drug delivery route.

Term " derived from " be used for representing the source (for example, this molecule can be derived from polynucleotides, polypeptide, and immortal cell line can be derived from any tissue, etc.) of molecule. If the first polynucleotides contain and the identical or essentially identical base-pair sequence in the second polynucleotides, its cDNA or a certain zone of its complementary series, if perhaps have above-mentioned sequence homogeny, then these first polynucleotides " derived from " the second polynucleotides. Therefore, if the first polynucleotides have (i) identical with the second sequence or essentially identical sequence, or (ii) and the polypeptide of this sequence have the sequence homogeny, then this first polynucleotide sequence " derived from " the second sequence.

If the first polypeptide (i) is coded by first polynucleotides derived from the second polynucleotides, or (ii) show with the second polypeptide to have above-mentioned sequence homogeny, then this first polypeptide " derived from " the second polypeptide. Therefore, if certain polypeptide (protein) (i) is encoded by certain ORF of SARS virus polynucleotides, or (ii) demonstrate with the SARS virus polypeptide and have above-mentioned sequence homogeny, then say this polypeptide (protein) " derived from " this specific SARS virus.

Polynucleotides and peptide molecule all can by physical method derived from SARS virus, perhaps, for example, can produce with recombination method or synthetic method according to known array.

The cell of cultivating or clone " derived from " if its initial cell or tissue that oneself has that gets of another cell or tissue. The nonrestrictive example that is used for obtaining the tissue of cell comprises skin, retina, liver, kidney, the heart, brain, muscle, intestines, ovary, breast, prostate, cancerous tissue, be subjected to tissue that one or more pathogen (such as virus, bacterium etc.) infect etc. Cell described here also can derived from other cell, include but not limited to the cell of primary culture, existing immortal cell line and/or other separation.

" antigen " refers to contain the molecule that one or more immune systems with stimulation of host produce the epi-positions that body fluid and/or cellular antigens-specificity reply (can be linearity or conformational epitope, or the two). This term can be replaced with term " immunogene " and use. Usually, an epi-position will comprise about 3-15, usually about 5-15 amino acid. The B-cell epitope contains 5 amino acid of having an appointment usually, but also can contain less such as 3-4 amino acid. The T-cell epitope such as the CTL epi-position, will contain at least 7-9 amino acid, and T-cell helper epitope contains at least about 10-20 amino acid. Usually, an epi-position will contain 7-15 the amino acid of having an appointment, such as 9,10,12 or 15 amino acid. Term " antigen " expression subunit antigen (antigen that namely separates and dissociate from natural complete organism of being combined with this antigen), and that kill, attenuation or deactivation bacterium, virus, fungi, parasite or other microorganism and tumour antigen, the interior part of born of the same parents that comprises the ectodomain of cell surface receptor and contain the T-cell epitope. Antibody, such as anti--idiotype antibody, or its fragment, and synthetic peptide mimic epitope (but analogue antigen or antigenic determinant) is also contained in the used here antigen definition. Similarly, (for example in gene therapy and dna immunization method) expressed the oligonucleotides of antigen or antigenic determinant or polynucleotides and is also contained in the antigen definition here in vivo.

" immune response " to certain antigen or composition is that the experimenter produces body fluid and/or cellullar immunologic response reaction to this antigen in the interested composition. For purposes of the present invention, " HI " refers to the immune response of antibody molecule mediation, comprises secretion property (IgA) or IgG molecule, and " cellullar immunologic response " is T-lymphocyte and/or other is from cell-mediated. An importance of cellular immunity relates to the antigentic specificity of cytolytic T-cell (" CTL ") and replys. CTL pair of peptide antigen that combines with protein that major histocompatibility complex (MHC) is coded in cell surface expression and offer has specificity. CTL helps to induce and promotes to destroy the cell that microorganism in the born of the same parents or dissolving are subject to this infected by microbes. The other method of cellular immunity relates to the antigentic specificity of helper T-cell and replys. " cellullar immunologic response " also refers to produce cell factor, chemotactic factor (CF) and other this quasi-molecule of activation T-cell and/or the generation of other leucocyte, comprises that those are derived from the molecule of CD4+ and CD8+T-cell. In addition, various leucocytes or endothelial cell can be induced the generation chemotactic factor (CF) in to the antigen-reactive that gives.

II. bacterin preparation

The present invention relates to be used for the treatment of or prevent the bacterin preparation of severe acute respiratory syndrome (SARS). Bacterin preparation of the present invention comprises deactivation (or killing) SARS virus, attenuation SARS virus, cracking SARS virus goods and the recombinant of one or more SARS virus antigens or the subunit prepara-tions of purifying. The present invention includes polypeptide and the polynucleotides of encoding SARS viral antigen and immunogenic fragments thereof. By viral vectors and/or virion, comprise virus-like particle (VLP), be of value to expression and the conveying of polynucleotides of the present invention.

A. deactivation (or kill) the SARS vaccine

The present invention includes the composition and the manufacture method thereof that contain deactivation (or killing) SARS virus. The deactivation of SARS virus composition can be used for preventative or therapeutic SARS virus vaccine. Preferred described deactivation of SARS virus vaccine combination comprises a certain amount of deactivation of SARS virus, and this amount was equivalent to before deactivation virus titer and is about every milliliter of 4-7log and has a liking for bacterial plaque and form unit (PFU) or 4-7log half tissue culture infective dose (TCID₅₀). More preferably virus titer is 4-11,7-11 or 9-11PFU or TCID before the deactivation₅₀ More preferably described deactivation of SARS virus vaccine combination comprises a certain amount of deactivation of SARS virus, and this amount is equivalent to that virus titer is about every milliliter of 5-9PFU or 5-9TCID before deactivation₅₀ In one embodiment, the PFU of the SARS virus of cultivation or TCID₅₀When results, be 6-8, be more preferably every milliliter of 7.5PFU or TCID₅₀ With regard to the concentration during with regard to virus harvest, PFU or TCID₅₀Be preferably 8-11, more preferably every milliliter of about 9PFU or TCID₅₀ Described vaccine combination contains the SARS virus antigen that is enough to produce the immune response amount in primate.

Deactivation known in the art or kill virus method be the ability of break virus mammalian cell-infecting. This method comprises chemical method or physical method. The chemical method of deactivation of SARS virus comprises following one or more agent treatment viruses with effective dose: detergent, formaldehyde, formalin, beta-propiolactone or UV light. Other chemical ablation method comprise with methylene blue, psoralen, carboxyl fullerene (carboxyfullerene) (C60) or their combination process. Other virus inactivating method is that this field is known, such as binary ethamine (binary ethylamine), acetyl group ethylene imine or radiation gamma.

For example, can be following concentration use formaldehyde, such as 0.1-0.02%, preferably 0.02-0.1%, more preferably 0.04-0.05%. Before or after the container results culture supernatant that is used for transmitted virus, in described culture supernatant, adding inactivator, can destroy or destroy cell before the results with the virus of release with Cell binding. In addition, inactivator can add after described culture supernatant has been frozen preservation and has thawed, or carries out with after removing the cell contamination thing in a step or multistep purifying. Yet, preferably after removing cell and cell fragment or after a step or the multistep purifying, add formaldehyde. Add after the formaldehyde, to contain virulent mixture transfers to culture vessel and cultivated 12 hours to 7 days under the room temperature of 20-30 ℃ or 33-37 ℃ according to appointment at refrigerated storage temperature (for example+2 to 8 ℃) or at higher temperature, wherein, selected temperature should adapt with the cultivation time. Preferably condition is, for example in+2 to 8 ℃ of lower cultivations 3-7 days (preferred 3-7 days), at room temperature cultivates 16 hours to 3 days (preferred 24-48 hour), or cultivates 12-36 hour under 35-37 ℃. If need to remove excessive formalin, can after finishing, inactivation process add sodium thiosulfate or the sodium pyrosulfite that waits mole or 1.5 times of molar concentrations (phase PARA FORMALDEHYDE PRILLS(91,95)).

For example, can following concentration use beta-propiolactone, such as 0.01-0.5%, preferred 0.5%-0.2%, more preferably 0.025-0.1%. Before or after the container results culture supernatant for transmitted virus, containing adding inactivator in the virulent described culture supernatant (viral material), can carry out or not destroy cell before the results with the step of release with the virus of Cell binding. In addition, inactivator can add after described culture supernatant has been frozen preservation and has thawed, or carries out with after removing the cell contamination thing in a step or multistep purifying. Beta-propiolactone is added viral material, control with NaOH (for example 1N NaOH), Tris buffer solution or sodium bicarbonate solution and can produce dysgenic pH oxytropism variation. Mixture is transferred to after another deactivation container, with inactivator-viral material mixture 4-37 ℃ of lower the cultivation a period of time, preferred 24-72 hour.

Other available inactivator is binary ethylene imine (BEI, binary ethyleneimine). With the bromine ethamine hydrobromide solution of 0.2 mole of equal-volume and 0.4 mole mixed about 37 ℃ of cultivations 60 minutes that are incorporated in of sodium hydroxide solution. The inactivator of gained cyclisation is the binary ethylene imine, and with volume ratio 0.5-4%, preferred 1-3% adds viral material with it. The viral material of deactivation is about 4-37 ℃ of lower the placement 24-72 hour, to stir. Cultivating the aseptic hypo solution that adds 1 mole of 20ml when finishing is neutralized to guarantee BEI.

In one embodiment, the present invention includes a kind of ablation method, this method be designed to make virus at utmost to be exposed to inactivator and make temperature sensitive SARS virus particle at elevated temperatures open-assembly time the shortest. The present invention includes a kind of ablation method, the method comprises makes virus contact 12-24 hour with inactivator (such as BPL) under refrigerated storage temperature, and the temperature that then raises was hydrolyzed any remaining inactivator in 3 hours. Preferred described refrigerated storage temperature is 0-8 ℃, more preferably from about 4 ℃. The temperature of preferred described rising is 33-41 ℃, more preferably from about 37 ℃. Infection virus remaining in every part of 10ml deactivation preparation is assessed as can be known, and the method can the deactivation initial cell be cultivated the SARS-CoV in the cutting, makes it below theoretical value of being down to 0.03 infectious unit/ml.

In permissive cell (tissue) culture (such as VERO), add dilution and undiluted inactivation of viruses material sample to detect the virus of any not deactivation. The passage of cultivation is repeatedly also used such as any situation that exists of checking SARS virus in cytopathic effect (CPE) and the antigen detection methods such as (such as by the fluorescein antibody coupling matter special to SARS virus). This detection can be determined complete inactivation of virus situation.

Before deactivation, in mammalian cell cultures, cultivate SARS virus. Cell culture can be the cell of adherent growth or be grown in cell in the suspension. Preferred cell is mammal source, but also can be from birds (for example chicken cell, such as chick-embryo cell (CEF cell)), amphibian, reptile, insect or fish. The mammal source of cell comprises, but be not limited to, the mankind or non-human primates cell are (for example, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), the HeLa cell, human diploid cell, rhesus macaque tire pneumonocyte (for example ATCC CL-160), human embryonic kidney cell's (293 cells, usually the Adenovirus Type 5 DNA that is sheared transforms), Vero cell (such as monkey kidney Vero cell), horse, ox (such as the MDBK cell), sheep, dog (dog kidney mdck cell for example, ATCC CCL34MDCK (NBL2) or MDCK 33016 are DSM ACC 2219 such as deposit number as described in the WO 97/37001), cat and mouse (for example hamster cell, such as BHK21-F, HKCC cell or Chinese hamster ovary cell (Chinese hamster ovary celI)), and can available from each stage of development, comprise for example adult, neonate, fetus or embryo.

In some embodiments, described cell is that infinite multiplication is (such as described in for example WO 01/38362 and the WO 02/40665 and to number the 96022940 PERC.6 cells that are preserved in ECACC, these two documents are included in this paper as a reference in full), or make any other cell type of its infinite multiplication with technology described here.

Used in preferred embodiments mammalian cell, this cell can be selected from and/or from following one or more non-limiting cell types: fibroblast (dermal cell for example, pneumonocyte), endothelial cell (sustainer cell for example, crown cell, pneumonocyte, vascular cell, the skin microvessel cell, the umbilical cord cell), liver cell, horn cell, immunocyte (for example, the T cell, the B cell, macrophage, the NK cell, dendritic cells), mammary cell (for example, epithelial cell), smooth muscle cell (for example, vascular cell, the sustainer cell, crown cell, arterial cell, uterine cell, the bronchus cell, cervical cell, the retina pericyte), melanocyte, nerve cell (for example, astroglia), prostatic cell (for example, prostatic epithelium, smooth muscle cell), nephrocyte (for example, epithelial cell, messangial cell, promixal tubular cell), bone cells (for example, the cartilage cell, osteoclast, Gegenbaur's cell), muscle cell (for example, sarcoblast, bone cells, smooth cell, the bronchus cell), liver cell, retinoblast and stroma cell. WO 97/37000 and WO 97/37001 (by including in full this paper in as a reference) described make can in suspension and serum free medium, grow, also can for the manufacture of with zooblast and the clone of replication-competent virus.

Preferred SARS virus of the present invention is cultivated in VERO cell or fetal rhesus kidney cell.

The condition of culture of above-mentioned cell type is described in detail in various publications, perhaps substitutive medium, additive and condition of culture can be bought by commercial sources, the for example catalogue of Cambrex Bioproducts (East Rutherford, NJ) and other document.

In some embodiments, be used for the host cell cultivation of method described here at the culture medium of serum-free and/or protein. When not containing the serum additive in human or animal source in the culture medium, be called serum free medium herein. Without protein be phalangeal cell in the situation that does not have protein, growth factor or other oroteins additive and non-serum proteins, occur propagation culture. The cell that is grown in this culture self contains protein naturally.

Known serum free medium comprises Iscove culture medium, Ultra-CHO culture medium (BioWhittaker) or EX-CELL (JRH Bioscience). The conventional culture medium that contains serum comprises Eagle minimal medium (BME) or minimum essential medium (MEM) (Eagle, Science, 130,432 (1959)) or the improved Eagle culture medium of Dulbecco (DMEM or EDM), wherein usually contain maximum 10% hyclones or similar additive. Randomly, can use minimum essential medium (MEM) (Eagle, Science, 130,432 (1959)) or the improved Eagle culture medium of Dulbecco (DMEM or EDM) that does not contain any serum additive. Protein-free culture medium, such as culture medium such as ProCHO 4CDM (BioWhittaker) or the SMIF 7 (Gibco/BRL Life Technologies) of PF-CHO (JHR Bioscience), specific chemical components, and mitogenesis peptide such as Primactone, Pepticase or HyPepTM (all from Quest International) or lactalbumin hydrolysate (Gibco is with other manufacturer) also are well known in the prior art. The special benefits of employing take plant hydrolyzed product as the medium additives on basis is to get rid of the pollution of virus, mycoplasma or unknown infectant.

The cell culture condition (temperature, cell density, pH value etc.) that is used for required purpose in very large range is different, with the demand that adapts to the used clone of the present invention and adapt to SARS virus.

The method of propagation SARS virus may further comprise the steps in the cell (such as mammalian cell) of cultivating: inoculate SARS virus in the cell of cultivating, the cell that infects is cultivated required time so that virus multiplication, incubation time will be decided by for example SARS virus titre or SARS virus antigen presentation (for example inoculation about 24-168 hour afterwards), and collect the virus of propagation. With virus (by PFU or TCID₅₀Mensuration) the cell ratio is 1: 10000-1: 10 ratio is inoculated SARS virus in the cell of cultivating. Can adopt lower proportion, for example 1: 500-1: 1, preferred 1: 100-1: 5, more preferably 1: 50-1: 10. SARS virus is added cell suspension or cell monolayer, and virus is adsorbed on the cell needs at least 60 minutes, usually is less than 300 minutes, preferred 25-40 ℃ 90-240 minute, more preferably 28-37 ℃, most preferably 33 ℃. With the cell culture (for example, individual layer) that infects by freeze-thaw or process to improve virus concentration in the culture supernatant of results by the enzyme effect. But then liquid or the freezing preservation of deactivation results.

Figure 26 A is the comparison of the Vero cell that infects of the SARS that cultivates when having and do not have hyclone (" FCS "). In brief, cultivate with the packing of Vero cell and in the T175 flask one day before infection. Second infects the 90% Vero cell monolayer that is paved with the SARS-SoV storage liquid (FRA strain, the 4th generation, accession number AY310120) that contains or do not contain 3%FCS. Adding FCS in cell culture medium shows almost not impact of viral yield.

Can about 0.0001-10, preferably 0.002-5, the more preferably cell of the infection multiplicity of 0.001-2 (" m.o.i. ") infection cultivation. Preferred, the m.o.i. infection cell with about 0.01. Viral yield relatively is shown in Figure 26 B under the different m.o.i. levels.

The cell of results infection in 30-60 hour after infecting. Preferred 34-48 hour harvesting after infection. More preferably 38-40 hour harvesting after infection. See Figure 26 C.

The method of purifying inactivation of viruses is known in the art, comprising following one or more, for example gradient centrifugation, ultracentrifugation, continuous-flow ultracentrifugation and chromatography are such as ion-exchange chromatography, size exclusion chromatography and liquid phase affinity chromatography. Other purification process comprises ultrafiltration and diafiltration. Referring to JP Gregersen " Herstellung von Virussimpfstoffen aus Zellkulturen ", Pharmazeutische Biotecnologie the 4.2nd chapters and sections (O.Kayser and RH Mueller compile) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000. Also can be referring to O ' Neil etc., " virus harvest and liquid phase affinity chromatography. Concentrate the method with purified virus " (Virus Harvesting and Affinity Based Liquid Chromatography.A Method for Virus Concentration and Purification), Biotechnology (1993) 11:173-177; Prior etc., " making improving one's methods of deactivation HIV-1 " (Process Development for Manufacture of Inactivated HIV-1), Pharmaceutical Technology (1995) 30-52; And Majhdi etc., " separation and the characteristic of the coronavirus of diarrhoea Elk calf " (Isolation and Characterization of a Coronavirus from Elk Calves with diarrhea) Journal of Clinical Microbiology (1995) 35 (11): 2937-2942.

The example that is suitable for other purification process of the present invention comprises that polyethylene glycol precipitation or ammonium sulfate surface precipitation are (referring to Trepanier etc., " with the concentrated human respiratory syncytial virus of ammonium sulfate, polyethylene glycol or Hollow Fiber Ultrafiltration " (Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration) Journal of Virological Methods (1981) 3 (4): 201-211; Hagen etc., " the polyethylene glycol precipitation hepatitis viruse is for the preparation of the optimization of high-purity inactivated vaccine VAQTA " (Optimization of Poly (ethylene glycol) Precipitation of Hepatitis Virus Used to prepare VAQTA, a Highly Purified Inactivated Vaccine) Biotechnology Progress (1996) 12:406-412; With Carlsson etc., " improve than infectiousness " (Purification of Infectious Pancreatic Necrosis Virus by An Iron Exchange Chromatography Increases the Specific Infectivity) Journal of Virological Methods (1994) 47:27-36 by anion-exchange chromatography purifying infectious pancreatic necrosis virus), and ultrafiltration and micro-filtration are (referring to Pay etc., Developments in Biological Standardization (1985) 60:171-174; Tsurumi etc., " being used for structure and strainability except the improved cuprammonium regenerated cellulose hollow fiber (improved BMM doughnut) of virus removal " (Structure and filtration performances of improved cuprammonium regenerated cellulose hollow fibre (improved BMM hollow fibre) for virus removal) Polymer Journal (1990) 22 (12): 1085-1100; With Makino etc., " with the concentrated liver retrovirus of regenerated cellulose hollow fiber BMM " (Concentration of live retrovirus with a regenerated cellulose hollow fibre, BMM), Archives of Virology (1994) 139 (1-2): 87-96.).

Preferably use purification by chromatography virus, for example ion-exchange chromatography. Chromatographic purifying can produce and contain in a large number virulent suspension. Interested viral product can interact with chromatography media by simple adsorption/desorption mechanism, and once-through operation can be processed bulk sample. There is not the impurity of affinity then to flow through pillar to adsorbent. Viral material can conc forms by wash-out.

Anion exchange resin preferred for the present invention comprises DEAE, EMI) TMAE. Preferred cationic ion-exchange resin can have the surface that sulfonic acid is modified. In one embodiment, first step is carried out ion-exchange chromatography, second step EMD-SO with containing reinforcing yin essence ion exchange resin (for example EMD TMAE)₃(cationic ion-exchange resin) purified virus. Can choose wantonly with metal and be further purified (referring to for example, WO 97/06243) in conjunction with affinity chromatography.

Being preferred for resin of the present invention is Fractogel^TMEMD. This synthetic methacrylate resin has the line polymer chain (so-called " tentacle ") of covalently bound length. This " tentacle chemicals (tentacle chemistry) " allows on the large quantity space come-at-able part to be combined with biomolecule and without spatial obstacle. This resin also has improved pressure stability.

Liquid phase affinity chromatography based on pillar is another kind of purification process preferred for the present invention. The example that is used for the resin of this purification process is Matrex^TMCellufineTM Sulfate (MCS). MCS is made of spherical (the diameter 45-105 μ m) cellulose matrix of rigidity, and its exclusion limit is 3000 dalton (the maximum molecules that its pore structure allows), at cellulosic 6 the sulfuric ester function bases that low concentration is arranged. Because functional part (sulfuric ester) relative altitude is disperseed, the cation exchange density that it provides is not enough so that most of soluble proteins are adsorbed in bead surface. Therefore, visible a large amount of protein have been washed from pillar in the typical virus liquid (cells and supernatant, for example pyrogen and most contaminating protein matter, and nucleic acid and endotoxin), and have realized the to a certain degree purifying to the virus of combination.

Rigidity high strength MCS pearl has resistance to compression trend. Thereby but the pressure/flow behavior of MCS resin allows high linear flow rate high speed processing, even if in large pillar, this makes its easy amplifying unit operation. MCS chromatographic purifying step has also been avoided too much product treatment and safety problem except the sterility that has improved security and product. Since endotoxin not with its combination, the MCS purification step can be fast and is not carried out depyrogenation with polluting. Gentle provides high power capacity and product yield in conjunction with elution requirement. Therefore, the MCS resin be a kind of simple, fast, concentrated, purifying and the depyrogenation method of efficient and cost-effective. In addition, the MCS resin is reusable.

The virus of deactivation also can be further purified by gradient centrifugation, the preferred density gradient centrifugation. For commercial-scale operation, continuous-flow sucrose gradient centrifugation is preferred. This method is widely used in the purifying antiviral vaccine and is be proficient in this field known by the technical staff (referring to JP Gregersen " Herstellung von Virussimpfstoffen aus Zellkulturen ", Pharmazeutische Biotecnologie the 4.2nd chapters and sections (O.Kayser and RH Mueller compile) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000. )

The density gradient centrifugation step can be carried out with laboratory scale or commercial-scale gradient centrifugation equipment. For example, swinging bucket rotor, angle rotor or vertical tube rotor are particularly suitable for laboratory scale and produce virus. Preferred described gradient centrifugation step is carried out with swinging bucket rotor. Such brick has sufficiently long passage length so that high-quality separation to be provided, especially to multicomponent sample. In addition, swinging bucket rotor has the waller effect that greatly reduces, and content need not be directed again between acceleration and deceleration period. Because the passage length that they are long, comparing to separate with angle rotor or vertical tube rotor needs the long period. Can control by refractometer the sucrose concentration of the sucrose solution of preparation.

Be used for density gradient centrifugation, such as the float bowl-type centrifuge tube, saccharose gradient can be before centrifugal form (continuously/linear) by gradient former. The volume that adds the sample on the swinging bucket rotor centrifuge tube inside gradient liquid is the function of the gradient liquid cross-sectional area of contact sample. If sample volume is excessive, then centrifuge tube does not have enough radical lengths to come each component in the effective separation multicomponent sample.

The sample volume that is used for swinging bucket rotor SW 28 is about every pipe 1-5ml (the pipe diameter is 2.54cm). By pipette sample is applied to gradient liquid top. Flush end and the tube wall of pipette are 45-60 ° of angle, about 2-3 millimeter above gradient liquid. Slowly inject sample and it is flow on the gradient liquid along tube wall. The all gradient fluid components of centrifugal rear recovery, method are carefully to insert gauge needle until the pipe end, and liquid is pumped into the falcon pipe begin to collect each component of 2ml from pipe.

The sucrose density gradient that is suitable for the density gradient centrifugation purification step comprises 0-60%, 5-60%, 15-60%, 0-50%, 5-50%, 15-50%, 0-40%, 5-40% and 15-40%. Preferably sucrose density gradient 15-40%, 5-40% or 0-40%.

Perhaps can adopt the discontinuous sucrose density gradient to carry out purifying. The Sucrose that the discontinuous sucrose densimetry adopts variable concentrations is from, overlapping layer. In an example, ground floor is 50% sucrose, covers the second layer 40% sucrose on it, covers the 3rd layer of 20% sucrose on the second layer, the 4th layer of 10% sucrose of the 3rd layer of upper covering, and the 4th layer of upper solution that contains virus to be purified that covers.

In one embodiment, the purification process of the virus of deactivation comprises first step chromatographic purifying and second step gradient centrifugation. The preferred first step comprises the liquid phase affinity chromatography, such as MCS. Preferred second step comprises with swinging bucket rotor and carries out density gradient centrifugation.

Other purification process of purifying deactivation of SARS virus comprises the employing nucleic acid-degrading agent, preferred nucleic acid digestive enzyme, as the nuclease with DNA enzyme and RNA enzymatic activity, or endonuclease, such as the endonuclease of serratia marcesens (Serratia marcescens), commodity are called Benzonase^TM, contain anionic functional group's membrane adsorbent (Sartobind for example^TM) or contain other chromatographic step (for example DEAE or TMAE) of anionic functional group's filler. Ultrafiltration/diafiltration and last aseptic filtration step also can be added into purification process.

Preferred purification process comprises the viral isolates with one or more nucleolysis enzyme treatment S ARS. These enzymes can be used to reduce the level of host cell nucleic acid in viral purification is processed. The nucleolysis enzyme that is used for cell culture is known in the art, comprises for example Benzonase^TM。

Processing virus with nucleolysis enzyme and inactivator can carry out or carry out with combination or simultaneous system successively. The preferred nucleic acid degradation agent was added into virus product before adding inactivator.

The virus product of purifying of the present invention is substantially free of the contaminated protein from cell or cell culture, preferred every microgram viral antigen contains and is less than about 1000,500,250,150,100 or the nucleus of 50pg, and preferred every dose contains and is less than about 1000,500,250,150,100 or the 50pg nucleus. More preferably the virus product of described purifying contains and is less than about 20pg, more preferably less than about 10pg nucleus. This field of detecting the method for host cell nucleic acid level in the virus sample is known. The approval of the management organization such as WHO or FDA or the standard method of recommending are preferred.

The present invention includes a kind of inactivated vaccine composition, wherein contain the SARS virus antigen that prevents effective dose, preferred furcella or its immunogenic fragments. Preferably there is more preferably 0.3-30mg antigen/agent in described SARS virus antigen with the concentration amount of 0.1-50 μ g antigen/agent. More preferably the antigen amount is about 15 μ g/ agent.

In one embodiment, adopted the SARS virus antigen of low concentration in the inactivated vaccine composition of the present invention. The vaccine of this low concentration can be chosen wantonly and contain adjuvant to cause the host to the immune response of antigen. In this " low dosage " vaccine, SARS virus antigen preferably exists with the concentration that is lower than 15 μ g antigen/agent, namely is lower than 10,7.5,5 or 3 μ g antigen/agent.

Inactivated vaccine goods of the present invention also contain stabilizing agent to keep the integrality of immunogenic protein in the inactivation of viruses goods. The stabilizing agent that is suitable for vaccine is known in the art, can comprise, for example, buffer solution, sugar, sugar alcohol and amino acid. Stable buffer solution the physiological pH scope should be transferred to, and phosphate buffer, Tris buffer solution, TE (Tris/EDTA), TEN (Tris/Nacl/EDTA) S and Earle salting liquid can be comprised. Stability sugar can comprise for example sucrose, glucose, fructose, glucan, sulfuric acid glucose and trehalose. Stability sugar alcohol can comprise, for example, xylitol, sweet mellow wine, sorbierite and glycerine. Be applicable to amino acid of the present invention and comprise, for example, Glu, arginine, cysteine and lysine, can be used for other stabilizing agent of the present invention and comprise tartaric acid, Pluronic F 68 and Tween 80.

The SARS virus separator that can be used for inactivation of viruses goods of the present invention can obtain and evaluation by above-mentioned any method. For example, the SARS separator can obtain from the bacterial plaque of having a liking for of clinical sample and purifying. The method of this isolated viral is known in the art.

Can adopt other purification process not contain for the preparation of the viral seed of vaccine guaranteeing, for example unwanted external reagent. In one embodiment, from the viral RNA of viral isolates can from virus, separate, purifying (and optional confirm by PCR or other method subsequently), then be introduced into suitable cell culture.

As an example of this technology, clinical virus sample is through having a liking for the bacterial plaque purifying and producing the capacity virus sample for analysis in vero cell propagation. The centrifugal cell fragment of removing in the supernatant. But Ultracentrifugation virus and virus group is resuspended in PBS then. Again after the centrifugal purification, contain virulent component with DNA enzyme (also can choose the RNA enzyme wantonly) processing. Then separate from this component and obtain viral RNA and it is transfected into host cell.

Embodiment 2 and 3 has exemplified a kind of method of complete S ARS virus of purifying deactivation, and the method adopts MCS chromatography purifying resin then to carry out density gradient ultracentrifugation.

The approaches and methods of immunity inoculation vaccine of the present invention will be described in detail in the chapters and sections below. Embodiment 4 and 5 provides the example that mouse is carried out immunity inoculation with deactivation of SARS virus of the present invention.

The B.SARS attenuated vaccine

The present invention includes a kind of composition that contains the attenuation SARS virus. This composition can be used as preventative or therapeutic SARS virus vaccine. The method that makes viral attenuation is known in the art. These methods with SARS virus at the cell of cultivating (for example comprise, mammalian cell cultures, preferred fetal rhesus kidney cell or Vero cell-see relevant narration of cultivating SARS virus in the A part) continuous passage is until the perform toxic attenuation of proof SARS virus in. The viral growth temperature can be can go down to posterity any temperature of attenuation of tissue culture. The perform toxic attenuation degree of SARS virus can be detected by the technical staff who is proficient in this field after one or many went down to posterity in cell culture. Attenuation refers to that here SARS virus virulence in human body reduces. The virus replication level that can show as the evidence of perform toxic attenuation reduces or the virulence in animal model reduces.

Other method of making the attenuation SARS virus be included in time good or " cold " temperature under in cell culture, go down to posterity virus and in the SARS virus genome, introduce the sudden change that makes perform toxic attenuation by random mutagenesis (for example mutagenesis) or direct mutagenesis. The method (this method also is suitable for usually for SARS virus) of preparation and generation attenuation REV vaccine is disclosed in, and for example, EP 0 640 128, U.S. Patent No. 6284254, U.S. Patent No. 5922326, U.S. Patent No. 5882651.

The attenuation derivative of SARS virus can be used the several method manufacturing, and for example, the introducing temperature-sensitive mutation goes down to posterity in by culture under " cold " temperature when being with or without chemical mutagen (for example 5 FU 5 fluorouracil). This acclimatization to cold process is included in about 20-32 ℃, preferably at about 22-30 ℃, most preferably goes down to posterity under about 24-28 ℃. Acclimatization to cold or attenuation can go down to posterity to introduce other growth restriction sudden change under the temperature that reduces gradually. Obtain at least part of condition that adopts that depends on of passage number safety, that immune attenuated virus is required. Virulence and immunocompetence to the SARS virus culture in animal (for example mouse, primate) are carried out the needed parameter of particular combinations that tissue culture and temperature can be easily determined in routine test. Usually attenuated vaccine is mixed with every dose maximum effectiveness about 10³-10 ⁶PFU or TCID₅₀Or more than.

Also can make by creating viral chimeras the attenuated virus vaccine of SARS-CoV, comprise the sequence from least two kinds of different coronavirus in the described chimera, wherein a kind of is SARS-CoV. For example, made a kind of from the first coronavirus (for example containing, mouse, ox, pig, dog, cat, birds coronavirus) the gene of coding non-structural protein and the viral chimeras of the gene (for example, furcella, E, M) of one or more coding structure albumen from SARS-CoV. Perhaps, this viral chimeras can contain the sequence of the human corona virus (for example, OC43,229E) from non-SARS-CoV and from the sequence of SARS-CoV. Chimeric coronavirus of the present invention can produce by the whole bag of tricks, for example comprise natural RNA is recombinated in eucaryon (such as mammal) cell, contain separately the RNA of parent coronavirus (for example after infecting) in the described eukaryotic, perhaps coming engineering to make up required viral chimeras (or its part) with the standard molecular biological technique known to the skilled of being proficient in this field clones as cDNA, then with they make infective virus (referring to for example, US 6593111 B2; Yount etc., 2003, Proc.Natl.Acad. Sci.USA 100 (22): 12995-13000). The chimeric attenuation phenotype of coronavirus described here is that the technical staff who is proficient in this field detects easily.

The virus of attenuation also can produce by the unessential ORF (ORF) of the one or more virus replications of deletion. Preferred these deletions occur in the genomic structural region, such as ORF 3a, 3b, 6,7a, 7b, 8a, 8b, 9b. Referring to, for example, Haijema BJ, Volders H, Rottier PJ.J Virol. (2004) 78 (8): 3863-71; And de Haan, C.A., P.S.Masters, X.Shen, S.Weiss and P.J.Rottier, " group-specific mouse coronavirus gene is nonessential; but by reverse genetics their deletions can be produced attenuation in natural host " and (The group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host) Virology (2002) 296:177-189. This zone in the coronavirus such as deletion SARS can be passed through, for example, reverse genetics method or " directed restructuring " realize, see for example Masters, P.S., " reverse genetics of maximum RNA virus " Adv.Virus Res. (1999) 53:245-264.

The method of purifying attenuated virus is known in the art, can comprise following one or more methods, for example gradient centrifugation and chromatography. Referring to Gregersen " Herstellung von Virussimpfstoffen aus Zellkulturen ", Pharmazeutische Biotecnologie the 4.2nd chapters and sections (O.Kayser and RH Mueller compile) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.

The C.SARS split vaccine

The present invention includes the composition and the manufacture method thereof that contain cracking SARS virus preparation. This composition can be used as preventative or therapeutic SARS virus vaccine.

The method of cracking enveloped virus is known in the art. The method of cracking enveloped virus is described in, and for example, WO 02/28422, fits into this paper in it as a reference, and particularly including wherein said decomposition agent and method. The method of cracking influenza virus is described in, for example, WO 02/067983, WO 02/074336 and WO 01/21151, they are included in this paper as a reference respectively in full.

Lytic virus is to use the decomposition agent of the concentration of breaking to break or the fragmentation intact virus, and this virus can be infectiousness (wild type or attenuation) or non-infectious (for example deactivation) virus. The fracture cause all or part of dissolving of virus protein, changed virus integrality.

Preferred described decomposition agent is nonionic or ionic surfactant. Therefore, cracking SARS virus preparation of the present invention also comprises at least a nonionic surface active agent or detergent. The example that is used for decomposition agent of the present invention comprises: bile acid and derivative thereof, nonionic surface active agent, APG or alkylthio glucosides and derivative thereof, acyl group sugar, sulfobetaines, betaine, polyoxyethylene alkyl ether, N; N-dialkyl group-glucamide (N, N-dialkyl-Glucamide), Hecameg, alkyl phenoxy polyethoxy ethanol, quaternary ammonium compound, sarcosyl, CTAB (cetrimonium bromide) or cetavlon (Cetavlon).

Preferred described ionic surfactant is cationic detergent. Be applicable to cationic detergent of the present invention and comprise the detergent that contains the compound with following formula:

Wherein,

R ₁、R ₂And R₃Can be identical or different, represent separately alkyl or aryl, or

R ₁And R₂And the nitrogen-atoms of their institute's combinations forms 5 or 6 yuan of heterocycles together, and

R ₃The expression alkyl or aryl, or

R ₁、R ₂And R₃Nitrogen-atoms with their institute's combinations forms undersaturated 5 or 6 yuan of heterocycles on the nitrogen-atoms,

R ₄The expression alkyl or aryl, and

X represents anion

The example of this cationic detergent has cetyltrimethyl ammonium salt, such as cetab (CTAB) and myristyl leptodactyline.

Be applicable to other cationic detergent of the present invention and comprise lipofectine, fat transfection amine (lipofectamine) and DOT-MA.

Be applicable to nonionic surface active agent of the present invention comprise following one or more: Octylphenoxy or Nonylphenoxy polyoxygenated alcohols (polyoxyethanol) (for example commercially available Triton series), polyoxyethylene sorbitan ester class (Tween series) and polyethenoxy ether class or have the ester class of following general formula:

                          O(CH ₂CH ₂O)n-A-R

Wherein, n is 1-50, A be a key or-C (O)-, R is C_1-50Alkyl or phenyl C_1-50Alkyl; And wherein two or more combination.

The present invention includes the method for making the SARS lytic virus, the method comprises that the decomposition agent that makes SARS virus contact capacity is with the break virus coating. Virus has been lost integrality and is made virus not have an infectiousness after the cracking. Just no longer be combined with complete virion in case virus envelope protein is destroyed, other virus protein just can dissolve wholly or in part, thereby with only partly is not combined with complete virion after cracking.

The method of making the SARS lytic virus also can comprise removes decomposition agent and part or most of viral lipid material. This process also comprises some distinctiveness filtration steps and/or other separating step, such as the various combinations of ultracentrifugation, ultrafiltration, band centrifugation and chromatographic step. This process also can be chosen wantonly and comprise inactivation step (as mentioned above), and this inactivation step can be carried out before or after cracking. Cleavage step can be in batches, continuous or semicontinuous mode is carried out.

SARS split-virus vaccine of the present invention can comprise structural proteins, membrane-bound fragment and membrane envelope albumen. Preferred SARS lytic virus goods of the present invention comprise at least half virus structural protein.

An example making the method for SARS lytic virus preparation may further comprise the steps:

(i) in the cell cultures such as MRC-5 cell (ATCC CCL-171), WI-38 cell (ATCC CCL-75), fetal rhesus kidney cell or vero cell, breed SARS virus (referring to the above narration of the relevant SARS virus cultivation of A part);

(ii) from cell culture, gather in the crops the material that contains SARS virus;

(iii) material of clarification results is to remove non-SARS virus material;

(iv) SARS virus of concentrated results;

(v) make complete SARS virus and non-viral separating substances;

(vi) in the density gradient centrifugation step with the complete SARS virus of suitable decomposition agent cracking; With

(vii) filter to remove unwanted material.

Above-mentioned steps should be carried out in order.

Clarification steps should be finished by the medium speed is centrifugal. Perhaps, filtration step can use for example film of 0.2 μ m.

Concentration step should adopt adsorption method, for example, uses CaHPO₄ Perhaps can adopt filtration, for example ultrafiltration.

Other separating step also can be used for method of the present invention. Other separating step is preferably band centrifugation to be separated, and can choose the employing saccharose gradient wantonly. Also can contain anticorrisive agent in the saccharose gradient to prevent growth of microorganism.

Cleavage step also can be carried out in saccharose gradient, wherein, contains decomposition agent in the described saccharose gradient.

The method also can comprise the aseptic filtration step, and this step can be chosen wantonly when processing finishes and carry out. One inactivation step is arranged before the preferred last filtration step.

The method for preparing SARS lytic virus preparation also can comprise with the DNA digestive ferment processes this virus formulation. These enzymes can be used to reduce the level of host cell DNA in the viral purification step. The DNA digestive ferment that is used for cell culture is known in the art, comprising, for example, Benzonase^_。

Can whenever carrying out in purification process and cracking processing with DNA digestive ferment treatment S ARS virus formulation. Yet preferably before using detergent, use DNA digestive ferment treatment S ARS virus formulation. More preferably before processing with cationic detergent such as CTAB, use the DNA digestive ferment treatment S ARS virus formulation of Benzonas and so on.

The method of purifying lytic virus is known in the art. Referring to JP Gregersen " Herstellung von Virussimpfstoffen aus Zellkulturen ", Pharmazeutische Biotecnologie the 4.2nd chapters and sections (O.Kayser and RH Mueller compile) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.

The present invention includes the split vaccine composition, wherein contain the SARS virus antigen that prevents effective dose, preferably its crack fragment or immunogenic fragments. The preferred 0.1-50 μ of the concentration of described SARS virus antigen g antigen/agent, more preferably 0.3-30 μ g antigen/agent. More preferably described antigen is about 15 μ g/ agent.

In one embodiment, in split vaccine composition of the present invention, used the SARS virus antigen of low concentration. The vaccine of this low concentration can be chosen wantonly and contain a kind of adjuvant to strengthen the host to the immune response of antigen. In this " low dosage " vaccine, the concentration of SARS virus antigen preferably is lower than 15 μ g antigen/agent, namely is lower than 10,7.5,5 or 3 μ g antigen/agent.

The D.SARS subunit vaccine

The present invention includes the composition that contains separative purifying SARS virus antigen or derivatives thereof. This composition also can contain one or more adjuvants.

SARS virus antigen can the SARS virus from be grown in cell culture in isolated or purified. Perhaps, the SARS viral antigen can produce with the methods known in the art restructuring.

Being used for SARS virus antigen of the present invention can make at many different expression systems, and these expression systems are known in the art; For example use the expression system of mammalian cell, baculoviral, bacterium and yeast. This expression system uses the polynucleotides of code book invention viral antigen usually. This sequence available standards Protocols in Molecular Biology obtains, comprising translating the amino acid sequence of listing here. Therefore, the present invention includes the polynucleotides of code book invention viral antigen. In addition, viral antigen of the present invention can be by chemical synthesis manufacturing (at least part of, as to be preferably integral body).

Insect cell expression system such as rhabdovirus system, is be proficient in this field known to the skilled, and is described in for example Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). The used materials and methods of the cell expression system of baculoviral/insertion can obtain by commercially available kit, comprising Invitrogen, and San Diego CA. Similarly, bacterium and mammalian cell cell expression system also can known in the artly be, and be described in the Butterworths such as " yeast genetic engineering " (Yeeat Genetic Engineering) (volume such as Barr, 1989), London.

Known many suitable host cells that can be used for said system. For example, mammal cell line is known in the art, comprise the immortal cell line available from American Type Culture Collection (ATCC), for example, but be not limited to, Chinese hamster ovary (CH0) cell, HeLa cell, baby hamster kidney cell (BHK) cell, MK cells (for example, Hep G2), Madin-Darby ox kidney (" MDBK ") cell and other cell. The mammal source of cell comprises, but be not limited to, the mankind or non-human primates cell are (for example, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), rhesus macaque tire pneumonocyte (for example ATCC CL-160), human embryonic kidney cell's (293 cells, usually the adenovirus type 5DNA that is sheared transforms), Vero cell (such as monkey kidney Vero cell), horse, ox (such as the MDBK cell), sheep, dog (dog kidney mdck cell for example, ATCC CCL34 MDCK (NBL2) or MDCK 33016, be DSM ACC 2219 such as deposit number as described in the WO 97/37001), cat and mouse (hamster cell for example, such as BHK21-F, HKCC cell or Chinese hamster ovary cell (Chinese hamster ovary celI)), and can available from each stage of development, comprise for example adult, neonate, fetus or embryo.

Similarly, the bacterial hosts such as Escherichia coli (E.coli), bacillus subtilis (Bacillus subtilis) and streptococcus also can be used for expression construction of the present invention. Be used for yeast host of the present invention and comprise saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), maltose Candida (Candida maltosa), Hansenula polymorpha (Hansenuala polymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Pichia guillerimondii, pichia pastoris phaff (Pichia pastoris), fission yeast (Schizosaccharomyces pombe) and Yarrowia lipolytica (Yarrowia lipolytica). The insect cell that is used for rhabdovirus expression vector comprises Aedes aegypti (Aedes aegypti), autographa california (Autographa californica), silkworm (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and broccoli looper (Trichoplusia ni).

The nucleic acid molecules stable integration that the range gene conveying technology that available this field is known will contain viral antigen of the present invention or antibody coding nucleotide sequence enters in the host cell gene group or remain on stable episome element in suitable host cell. Referring to, for example, U.S. Patent No. 5,399,346.

According to selected expression system and host, the host cell that expression vector is transformed is bred and the generation desired molecule. Then separate protein and the purifying of expressing from host cell. If expression system is secreted into protein in the growth medium, then can be from culture medium direct purification product. If it is not secreted then can from cell lysate, separate. Selecting suitable condition of culture and recovery method is be proficient in this field known to the skilled.

The present invention includes the composition of the SARS virus antigen or derivatives thereof that contains isolated or purified. The present invention also comprises the composition of the SARS virus antigen or derivatives thereof that contains at least two kinds of isolated or purifieds, and these two kinds of materials are by common purifying or by separately purifying and then merging. In one embodiment, described SARS virus antigen is furcella (S) albumen. Again in another embodiment, described SARS virus antigen is nucleocapsid (N) albumen, film (M) glycoprotein or coating (E) albumen. The purity of SARS virus antigen is greater than 75% (for example, 78%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98%) in the preferred described composition.

The present invention includes a kind of vaccine combination, wherein contain the SARS virus antigen that prevents effective dose, preferred furcella or its immunogenic fragments. The concentration of described SARS virus antigen is preferably 0.1-50 μ g antigen/agent, more preferably 0.3-30 μ g antigen/agent. Again preferably, the about 15 μ g/ agent of antigen.

In one embodiment, in vaccine combination of the present invention, used the SARS virus antigen of low concentration. The vaccine of this low concentration can be chosen wantonly and contain a kind of adjuvant to cause the host to the immune response of antigen. In this " low dosage " vaccine, the concentration of SARS virus antigen preferably is lower than 15 μ g antigen/agent, namely is lower than 10,7.5,5 or 3 μ g antigen/agent.

Following examples have exemplified the method for preparing SARS virus furcella (S) protein subunit vaccine.

Available several different methods is separated and purifying SARS virus S antigen from multiple source, and these methods include, but not limited to express S antigen in the eukaryotic of cultivating (for example mammalian cell, such as VERO, CHO) or bacterium (for example Escherichia coli). Expression can realize by the whole bag of tricks, for example, express from the cell culture or the cell culture supernatant that are infected by SARS virus, from the DNA expression cassette of encoding SARS virus S protein (for example, operability is connected in the rna plymerase ii promoter of SARS virus S gene) cultured cell of stable conversion expresses, from the reproducible of encoding SARS virus S protein or not replication-competent virus expression vector (for example, adenovirus vector, poxvirus vector, α viral vectors, retroviral vector) cultured cell that infects expresses, and so just do not need to use infectiousness SARS virus.

1. produce the SARS subunit vaccine from the SARS virus culture

SARS virus can be grown in the mammalian cell of cultivation, in the Vero cell, then obtains from the cell separation of cultivating. Then can make SARS virus antigen, separate with SARS virus such as the S protein dissolution, further separate again and purifying.

Among one embodiment, can make SARS virus according to the method for SARS inactivated vaccine embodiment, then be further purified required SARS antigen with the known technology in this field from end-product, such as spike protein.

Among another embodiment, the SARS subunit vaccine can be made in accordance with the following methods. Required mammal cell line is produced SARS virus on the microcarrier bead in large-scale controlling fermentation tank. For example, be 10 with concentration⁵The vaccine level African green monkey kidney cell (Vero cell) of cell/mL adds 60-75 and rises CMRL 1969 culture mediums, and pH 7.2, in 150 liters of bioreactors that contain 360 gram Cytodex-1 microcarrier beads, and stirs 2 hours. Add CMRL1969 to 150 liters of cumulative volumes. Adding hyclone (FBS) to ultimate density is 3.5%. Adding glucose to ultimate density is 3.0 g/L, and adding glutamine to ultimate density is 0.6g/L. Control dissolved oxygen, pH, stirring and temperature, monitoring Growth of Cells, glucose level, lactate levels and glutamine level. (usually at 3-4 days, density reaches about 1.0-2.5 * 10 when cell is in logarithmic phase⁶Cell/mL), pour out the culture medium in the fermentation tank and add 120 liters of CMRL 1969, pH 7.2 (without FBS) then stirs culture 10 minutes. Emptying and filling repeats once usually, but also can repeat nearly 3 times. Behind the washed cell, emptying fermentation tank also adds 50 liters of CMRL 1969 that contain 0.1% (v/v) FBS. Infection multiplicity (m.o.i) with 0.001-0.01 is added the SARS virus inoculum. Can add trypsase to promote effective infection. Add the CMRL 1969 contain 0.1%FBS to final volume be 150 liters. 34 ℃ of Continuous Cultivation. One time fermentation can obtain a kind of virus harvest thing, after infection 2-7 days usually. One time fermentation also can obtain the Multiple harvests thing.

Available following the whole bag of tricks separates and purifying S albumen. For example, collect the SARS virus envelope protein of dissolving from the efflux that contains S albumen of ion-exchange chromatography outflow; This efflux is loaded onto on the hydroxyapatite matrix, and from hydroxyapatite matrix selective elution S albumen. The S albumen of selective elution can be further concentrated by the mobile ultrafiltration of tangent.

Perhaps, can separate by the following method and purifying: collect the SARS virus envelope protein of dissolving from the efflux that contains S albumen of ion-exchange chromatography outflow; Efflux is loaded onto on the hydroxyapatite matrix and collects the efflux that contains S albumen, from hydroxy phosphorus Calx matrix efflux, selectively remove the used detergent of dissolving step, to obtain separating and the S albumen of purifying. The S albumen of separation and purifying subsequently can be by the mobile ultrafiltration concentration of tangent. Above available the described nucleic acid-degrading agent of deactivation part process to remove separate and the S albumen of purifying in contaminant nucleic acid. Preferred described nucleic acid-degrading agent is nuclease, such as Benzonase.

The S albumen of separation and purifying can be used for gel filtration medium also subsequently from wherein collecting S albumen with the separated from contaminants with S albumen and other molecular weight.

Perhaps, separation and purifying can be finished by the following method: S albumen is loaded onto the first Ion Exchange Medium, allows pollutant by this medium, from the first Ion Exchange Medium wash-out S albumen, with the separated from contaminants of S albumen and other molecular weight. The S albumen of wash-out is loaded onto the second Ion Exchange Medium, and makes pollutant pass through the second Ion Exchange Medium. Subsequently from wash-out S albumen wherein to obtain separating and the S albumen of purifying. The S albumen of wash-out can be by the mobile ultrafiltration concentration of tangent.

Perhaps, can be fit to can be used as from the cell lysate preparation of infecting the SARS virus S albumen of the immunogenic basic purifying of subunit vaccine preparation, for example dissolve infected cell with the non-sex change detergent buffer solution that contains 1%Triton X-100 and dexycholate. Cell lysate obtains S albumen by centrifugal clarification and by immunoaffinity purification from the cell lysate purifying. Produce the monoclonal antibody of anti-S albumen, make itself and pearl coupling also fill post with these pearls. To be applied to by the cell lysate that SARS infects this post, and wash this post with the PBS that contains 0.1%Triton X-100. Be attached to the protein 0.1M glycine on the post, pH 2.5,0.1%Triton X-100 wash-out. With the sample of for example Trix buffer elution and analyze the wherein existence of protein. Merging contains the component of protein and dialyses with PBS.

As mentioned above, the present invention includes the SARS virus S albumen of separation and purifying. Among one embodiment, this Virus culture in vaccine level clone, on the Vero cell, is gathered in the crops the virus of cultivating. Filter the virus harvest thing, then apparatus has the diafiltration of required molecular cut off to carry out the mobile ultrafiltration concentration of tangent. Can be with the virus harvest concentrate centrifugal and supernatant discarded. Then from centrifugal sediment, extract the S albumen of dissolving with detergent, for example, precipitation is resuspended in the volume of the extremely initial results concentrate of Extraction buffer that contains detergent (such as non-ionic detergent, comprising TRITON X-100).

Centrifugal removing after the undissolved protein is by chromatographic purifying S protein extract. Can first extract be added to ion exchange column, such as TMAE-fractogel or the S-fractogel post of balance, the S protein stream be crossed and impurity is retained on the pillar.

Then efflux is loaded onto on the hydroxyapatite column of overbalance, makes the S protein combination make pollutant pass through post to matrix. Then use the S albumen of suitable eluent elution of bound from the pillar. Can further process the S protein solution of gained purifying to improve its purity. At first available have the film of required molecular cut off by the mobile ultrafiltration concentration eluent of tangent. Filtrate is contacted with precipitating proteins with the polyethylene glycol with desired molecule amount (for example about 6000-8000). Centrifugal and supernatant discarded can be resuspended in sediment PBS afterwards and dialyse to remove polyethylene glycol. At last can be with the S protein solution aseptic filtration through dialysis. Liquid through aseptic filtration can be adsorbed on the alum. If necessary, the commitment in purification process carries out polyethylene glycol precipitation and resuspended purification step.

Perhaps, after cultivating and gathering in the crops virus, reclaim SARS virus, for example obtain concentrate with PEG precipitation or tangential flow filtration. Make virus contact to dissolve S albumen with detergent. Reclaim supernatant to be further purified S albumen and to discard insoluble protein after centrifugal.

Supernatant is loaded onto ion exchange column, and such as TMAE-fractogel or S-fractogel post, this post has passed through appropriate balance so that S albumen is retained on the pillar. Under suitable condition, S albumen is eluted from ion exchange column. Then make eluent pass through solvent resistant column, such as Sephacryl S-300 post, with the separated from contaminants with S albumen and other molecular weight. Available hydroxyapatite column replaces the Sephacryl post.

Can be from wash-out S albumen on the pillar to obtain the S protein solution of purifying. Eluent can be with the film with required molecular cut off by the mobile ultrafiltration concentration of tangent. But concentrated then aseptic filtration of S protein solution.

Perhaps, the virus harvest thing can pass through ultrafiltration concentration, and can carry out initial purification step to concentrated virus harvest thing, for example, and by gel permeation chromatography, polyethylene glycol precipitation or sulfuric acid Cellufine chromatography. Then the virus of available detergent-treatment purifying is with dissolving S albumen. Supernatant can be loaded onto ion exchange column after the dissolving S albumen, such as sulfuric acid Cellufine chromatographic column, the overbalance of this post so that protein bound to pillar and pollutant is flow through. Similarly, can replace sulfuric acid Cellufine post with TMAE-fractogel or S-fractogel post. Also these two pillars can be combined into the order purification step. S albumen is eluted to obtain the protein solution of purifying from pillar. Then can concentrate this solution by the mobile ultrafiltration of tangent and diafiltration with the film with required molecular cut off.

Specifically, in a kind of method of purifying S albumen, the virus harvest concentrate in 4 ℃ at 28,000 * g centrifugal 30 minutes. Supernatant discarded also is resuspended in the Extraction buffer that contains 10mM Tris-HCl (pH 7.0), 150mM NaCl, 2% (w/v) Triton X-100 to original results concentrate volume with precipitation. Add Pefabloc to ultimate density be 5mM. Suspension was at room temperature stirred 30 minutes. The supernatant that contains solubility S albumen under 4 ℃ with centrifugal 30 minutes of 28,000 * g so that its clarification. The TMAE-Fractogel post is with 10mM Tris-HCl (pH 7.0), 150mM NaCl (wherein containing 0.02%Triton X-100) balance. The Triton X-100 supernatant that will contain solubility S albumen is directly loaded onto the TRAE-Fractogel post. Collect the 10mM Tris-HCl of total application of sample volume and 2 times of column volumes, pH 7.0,150mM NaCl (containing 0.02%Triton X-100). The TMAE-Fractogel efflux 10mM Tris-HCl that will contain S albumen, 3 times of pH 7.0 (containing 0.02%Triton X-100) dilutions.

Hydroxyapatite column 10mM Tris-HCl, pH 7.0,50mM NaCl, 0.02%Triton X-100 balance. With the 10mM Tris-HCl of 2 times of column volumes, pH 7.0 behind the adding TMAE efflux, 50mM NaCl, and the 5mM sodium phosphate of 0.02%Triton X-100 and 4 times of column volumes, pH 7.0,1M NaCl, 0.02%Triton X-100 washs this post. Protein is with the 20mM sodium phosphate of 4 times of column volumes, and pH 7.0,1M NaCl, 0.02%Triton X-100 wash-out. Collect each compound mensuration antigen concentration according to A280 and protein content. The S albumen of purifying comes ultrafiltration with 300 kDa NMWL films by the mobile ultrafiltration of tangent.

2. the SARS subunit vaccine is made in restructuring

As mentioned above, SARS virus protein can be made by recombinant expressed. Be fit to recombinant expressed host cell and comprise bacterium, mammal, insect, yeast etc. Recombinant expressedly can be used to make total length SARS albumen, its fragment or its fusion product.

Fusogenic peptide can be used to be convenient to recombinate the SARS Protein expression and purification. For example, adding labelled protein in SARS antigen to be expressed expresses as the fusion that contains labelled protein and SARS antigen and helps restructuring to make the SARS polypeptide. This labelled protein helps protein purification, detection and the stability expressed. Be applicable to labelled protein of the present invention comprise poly arginine mark (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin binding peptide, cellulose binding domain, SBP-tag, chitin in conjunction with territory, glutathione s-transferase mark (GST), lactose in conjunction with albumen, tanscription termination antitermination factor (transcription termination anti-terminiantion factor) (NusA), escherichia coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). Preferred labelled protein comprises His-tag and GST. Can be referring to Terpe etc. about discussing in detail of labelled protein, " labelled protein merges summary: from molecule and biochemical basis to commercial system " (Overview of tag protein fusions:from molecular and biochemical fundamentals to commercial systems), Appl Microbiol Biotechnol (2003) 60:523-533.

After the purifying, labelled protein can be chosen wantonly from the fusion of expressing and remove, and namely shears enzyme by the known specificity in this field and processes. Normally used protein comprises enterokinase, marmor erodens (TEV), fibrin ferment and the Xa factor.

Therefore, the present invention also comprises the SARS virus subunit vaccine that contains fusion. Preferred described fusion comprises the first amino acid sequence by the SARS virus polynucleotide sequence coding. The SARS virus polynucleotide sequence of described the first amino acid sequence of encoding comprises one or more SARS virus polynucleotide sequence and fragments thereof of identifying in this specification.

Described fusion can comprise amino acid sequence or its fragment of SARS virus protein. Described SARS virus albumen is selected from one or more in the following SARS virus albumen: P28, P65, Nsp1, Nsp2 (3CL protease), Nsp3, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8s Nsp9 (RNA polymerase), Nsp10 (helicase), Nsp11, Nsp12, Nsp13, spike protein, Orf3, Orf4, envelope protein, stromatin, Orf7, Orf8, Orf9, Orf10, Orf11, nucleocapsid protein and Orf13.

In one embodiment, described fusion comprises the first amino acid sequence that contains SARS virus antigen or its fragment. Described SARS virus amino acid sequence can contain one or more T-epitope sequences of identifying above.

Preferred described fusion comprises amino acid sequence or its fragment of SARS virus spike protein. The spike protein specific fragment that can be used for fusion comprises S1 domain and S2 domain. Other spike protein fragment that can be used for fusion comprises the regional of S1 and S2 domain, comprises that receptor binding domain, the oligomerization zone of S2 domain, the leucine zipper district of S2 domain, the film anchor region of S2 domain, the hydrophobic domains zone of S2 domain, the S2 domain of S1 domain is rich in the zone of cysteine and the cytoplasmic tail territory (referring to Figure 19) of S2 domain. The amino acid sequence of the spike protein corresponding with these zones can be identified by the technical staff who is proficient in this field, comprises such as using the previous function prediction that proposes among the application (the coiled coil zone of the transbilayer helix of prediction, the terminal signaling zone of the N-of prediction, prediction etc.) and carrying out homology relatively (referring to Fig. 4 F and 5) with the sequence of other known baculoviral.

Described fusion also can comprise the second amino acid sequence. Described the second amino acid sequence can include the peptide sequence that helps S protein expression or purifying, and one of preferred this flag sequence is above being discussed. Perhaps, described the second amino acid sequence can comprise the second amino acid sequence of SARS virus. Perhaps, described the second amino acid sequence can comprise the amino acid sequence of other virus or bacterium (virus or the bacterium that comprise one or more I Partial Characterizations below).

Described the second amino acid sequence can comprise the amino acid sequence of other Respirovirus. But described the second amino acid sequence chelating is selected from the amino acid sequence of lower group virus: coronavirus, influenza virus, rhinovirus, parainfluenza virus (PIV), Respiratory Syncytial Virus(RSV) (RSV), adenovirus and super Pneumovirinae.

In one embodiment, described the second amino acid sequence can comprise the amino acid sequence of adjuvant, and described adjuvant comprises the adjuvant of one or more I Partial Characterizations below.

In one embodiment, the present invention includes amino acid sequence or its fragment that fusion comprises the SARS virus spike protein. Described fusion also can comprise the second amino acid sequence and adjuvant, and described the second amino acid sequence contains the amino acid sequence that is selected from the second SARS virus albumen, non-SARS virus albumen, bacterioprotein.

(a) bacterial expression of SARS subunit vaccine

In one embodiment, bacterial host cell is used to recombinant expressed SARS virus albumen. Be applicable to bacterial host cell of the present invention and comprise, for example, Escherichia coli (E.coli), bacillus subtilis (Bacillus subtilis) and streptococcus.

SARS virus albumen can be modified to be conducive to bacterium recombinant expressed. Specifically, the SARS spike protein can be modified to be conducive to spike protein and is transported to the bacterial host cell surface.

The applicant has disclosed between SARS virus spike protein and the Neisseria meningitidis NadA albumen strong structural homology. These two kinds of protein all have terminal spherical " head " domain (amino acid 24-87) of N-, very tend to form the alpha helical region territory (amino acid 88-350) of centre of coiled coil structure and the C-end film anchoring structure territory (the amino acid 351-405 of NadA) that is formed by 4 both sexes cross-film β chains. In addition, there is a leucine zipper motif in the coiled coil section. Referring to Figure 19, wherein show the structure of SARS spike protein, Comanducci etc., " vaccine candidate object that NadA-Neisseria meningitidis is new " (NadA, a Novel Vaccine Candidate of Neisseria meningitidis), J.Exp.Med.195 (11): 1445-1454 (2002). In addition, the leucine zipper motif of NadA is present in the coiled coil section. NadA albumen also forms the oligomer (corresponding to three or four monomers) that is exposed to the surface that is anchored on the meningococcus adventitia of HMW.

As NadA during at expression in escherichia coli, full length protein assembling or be anchored to the oligomer of Escherichia coli adventitia, the assembling approach of the protein that exists in this and the meningococcus is similar. Then the NadA protein that lacks the film anchoring structure territory of prediction be secreted in the culture supernatant. The protein of this secretion is soluble, and still is organized into trimeric form.

Therefore, the present invention includes a kind of fusion, wherein comprise amino acid sequence or the second amino acid sequence of its fragment and bacterium adhesion protein or the fusion of its fragment of SARS virus spike protein. Preferred described adhesion protein is selected from NadA, YadA (from enteron aisle pathogenicity Yersinia) and UspA2 (from Moraxella catarrhalis (Moraxella catarrhalis)). Other NadA sample albumen comprises saa gene (STEC) that the large virulence plasmid of outer membrane protein 100, the strain of colon bacillus shiga toxigenic bacterium of the serum resistant protein DsrA of bacillus ducreyi (Haemophilus ducreyi), colibacillary immunoglobulin-binding proteins EibA, C, D and F, actinobacillus actinomycetem comitans (Actinobacillus actinomycetemcomitans) is carried and the various bacterium adhesion proteins described in the UK Patent Application No.0315022.4 (on June 26th, 2003 submitted to, fitted into this paper in it as a reference).

Preferred described adhesion protein comprises NadA or its fragment.

This fusion can be used to promote SARS surface antigen immunogenicity part, recombinant expressed such as furcella. These fusion construct can make SARS S1 and/or S2 domain take native conformation. These fusions also can form dimer or tripolymer by oligomerization, and S1 and/or S2 domain are combined, and take the conformation of natural SARS spike protein. In addition, these expression constructions help the SARS spike protein to be exposed to the surface.

Fusion of the present invention preferably contains the leader peptide of the preferred NadA of NadA sample albumen, the polypeptide in spike protein immunogenicity " head " zone and the handle zone of NadA sample albumen or spike protein. When expressing and process fusion, can excise or remove, such as one or more amino acid in leader peptide or film anchoring structure territory.

The handle zone helps the oligomerization of expressing protein. Randomly, fusion of the present invention also can comprise the anchor zone of NadA sample albumen. This anchor zone is so that the fusion grappling of expressing and be assembled to the bacterial cell surface.

Fusion of the present invention contains following construction:

(i) NadA leader peptide (also can choose front 6 amino acid of containing ripe NadA albumen wantonly with the processing that is conducive to leader peptide and the Appropriate maturation of protein) is furcella S1 domain afterwards. The amino acid/11-29 that preferred this construction contains NadA is (corresponding with front 6 amino acid of NadA leader peptide and ripe NadA albumen, list as shown in figure 22 and hereinafter), thereafter the amino acid/11 4-662 that is the SARS virus spike protein is (corresponding with the S1 domain, see Figure 19 and SEQ ID NO:6042, and list below). Specifically, construction (i) comprises SEQ ID NO:7302.

(ii) NadA leader peptide (also can choose front 6 amino acid of containing ripe NadA albumen wantonly with the processing that is conducive to leader peptide and the Appropriate maturation of protein) is furcella S1 domain afterwards, is handle and the anchor membrane structure territory of NadA afterwards. The amino acid/11-29 that preferred this construction contains NadA is (corresponding with front 6 amino acid of NadA leader peptide and ripe NadA albumen, list as shown in figure 22 and hereinafter), thereafter the amino acid/11 4-662 that is the SARS virus spike protein is (corresponding with the S1 domain, see Figure 19 and SEQ ID NO:6042, and list below), be the amino acid 88-405 (corresponding with handle and anchor membrane structure territory) of NadA afterwards. Specifically, construction (ii) comprises SEQ ID NO:7303.

(iii) NadA leader peptide (also can choose front 6 amino acid that contain ripe NadA albumen wantonly) is SARS viral spikes S1 domain afterwards, is NadA handle structure territory afterwards. Preferred this construction contains the amino acid/11-29 of NadA, is thereafter the amino acid/11 4-662 (corresponding with the S1 domain) of SARS virus spike protein, is the amino acid 88-350 (corresponding with handle and anchor membrane structure territory) of NadA afterwards. Specifically, construction (iii) comprises SEQ ID NO:7304.

(iv) NadA leader peptide (also can choose front 6 amino acid that contain ripe NadA albumen wantonly) is SARS virus furcella S1 and S2 domain (the cross-film district that does not comprise supposition) afterwards, is the anchor structure territory of NadA afterwards. Preferred this construction contains the amino acid/11-29 of NadA, that the amino acid/11 4-1195 of SARS virus spike protein (corresponding to S1 and S2 thereafter, the cross-film district that does not comprise supposition), be afterwards the amino acid 351-405 (corresponding to NadA anchor structure territory) of NadA. Specifically, construction (iii) comprises SEQ ID NO:7305. Perhaps, NadA anchor structure territory can comprise the amino acid 332-405 of NadA.

(v) NadA leader peptide (also can choose front 6 amino acid that contain ripe NadA albumen wantonly) is SARS virus furcella S1 and S2 domain (the cross-film district that does not comprise supposition) afterwards. Preferred this construction contains the amino acid/11-29 of NadA, is thereafter the amino acid/11 4-1195 of SARS virus spike protein. Specifically, construction (v) comprises SEQ ID NO:7306.

In each of construction (i)-(v), front 23 amino acid are NadA leader peptides, and the GS dipeptides among the residue 679-680 inserts from restriction endonuclease sites.

At construction (i), (ii) with (iii), NadA " head " is substituted by furcella S1 domain, and this fusion is anchored to respectively on the Escherichia coli adventitia or is secreted in the culture supernatant. At construction (iv) with (v), " head " of NadA and " handle " domain are substituted by S1 and S2 furcella domain; At this moment, these two fusions are anchored to respectively on the Escherichia coli adventitia or are secreted in the culture supernatant.

Therefore, the present invention also comprises and contains SARS virus spike protein amino acid sequence or its fragment and bacterium adhesion protein the second amino acid sequence or its segment composition albumen. Preferably the amino acid corresponding with " head " of adhesion protein is by the amino acid replacement corresponding to SARS virus furcella S1 domain. Perhaps, the amino acid corresponding with " head " and " handle " domain of bacterium adhesion protein is by the amino acid replacement corresponding to SARS virus spike protein S1 and S2 domain.

As mentioned above and shown in Figure 19, the S1 domain of spike protein is accredited as spherical receptors bind " head " zone. The S1 domain of spike protein should comprise the amino acid of the about 14-662 of SEQ ID NO:6042 position. The S1 domain can comprise from N-end or C-stub area has removed some amino acid whose short amino acid sequences. Preferably having 3,5,7,9,13,15,20 or 25 amino acid is removed from N-end or C-stub area. The S1 domain also comprises the amino acid sequence that has the sequence homogeny with the S1 zone of SEQ ID NO:6042. An example of S1 domain is SEQ ID NO:7307.

With shown in Figure 19, the S2 domain of spike protein is accredited as " handle " zone as mentioned above. Should " handle " district inclusion oligomerization domain regional, the film anchor region in zone, leucine zipper motif, hydrophobic domains zone, domain zone and the cytoplasmic tail territory of being rich in cysteine. The S2 domain of spike protein should not comprise the cross-film district, and comprises the amino acid of the about 663-1195 of SEQ ID NO:6042 position. The S2 domain can comprise from N-end or C-stub area has removed some amino acid whose short amino acid sequences. Preferably having 3,5,7,9,13,15,20 or 25 amino acid is removed from N-end or C-stub area. The S2 domain also comprises the amino acid sequence that has the sequence homogeny with the S2 zone of SEQ ID NO:6042. An example of S2 domain (not comprising the cross-film district) is SEQ ID NO:7308.

An example of above-mentioned NadA albumen is SEQ ID NO:7309. As mentioned above, the targeting sequencing that is used for the NadA of fusion should comprise front 29 amino acid of NadA (about 6 amino acid that comprise targeting sequencing and NadA noggin). The example of this targeting sequencing is listed among the following SEQ ID NOS:7310 and 7311. Fusion can use to contain from N-end or C-stub area has removed some amino acid whose short amino acid sequences. Preferably having 1,2,3,4 or 5 amino acid is removed by N-end or C-stub area from this sequence. The targeting sequencing that is used for fusion also can comprise the amino acid sequence that has the sequence homogeny with SEQ ID NO:7310 or SEQ ID NO:7311. Preferred described targeting sequencing comprises SEQ ID NO:7311.

Randomly, this fusogenic peptide comprises front approximately 6 amino acid of ripe NadA albumen, is beneficial to the processing of leader peptide and the Appropriate maturation of protein. Front 6 amino acid whose one examples of ripe NadA albumen are SEQ ID NO:7312.

As mentioned above, be used for the handle of NadA of fusion and the amino acid that the anchor sequence should comprise the about 88-405 of NadA position. An example that comprises the amino acid sequence in NadA handle and anchor zone is SEQ ID NO:7313. An example that contains the amino acid sequence in NadA handle zone (not containing the anchor zone) is SEQ ID NO:7314. An example that contains the amino acid sequence in NadA anchor zone is SEQ ID NO:7315. The fusion of use handle (and/or anchor) sequence comprises from N-end or C-stub area has removed some amino acid whose short amino acid sequences. Preferably having 1,2,3,4,5,6,7,8 or 9 amino acid is removed by N-end or C-stub area from this sequence. The targeting sequencing that is used for fusion also can comprise the amino acid sequence that has a sequence homogeny with 7313.

Fusion of the present invention (comprise above-mentioned those) can according to, for example, the following methods preparation. The Oligonucleolide primers that available following table is listed is by pcr amplification individual chip (such as above-mentioned zone). (S1L represents the spike protein that merges with the leader peptide of NadA; S2 represents to contain or do not contain the handle zone of the spike protein of terminator codon). Designed oligonucleotides according to the dna sequence dna of Neisseria meningitidis (N.meningitides) B 2996 bacterial strain NadA and the dna sequence dna of SARS virus separator FRA1 furcella. Every kind of oligonucleotides comprise a restriction site as tail so that the clone is inducted among the expression vector pET21b.

SEQ ID NO: restriction site

S1L        For        7316        NdeI

S1L        Rev        7317        BamHI

S2             For    7318            BamHI

S2             Rev    7319            HindIII

S2-terminal point Rev 7320 XhoI

NadA ₈₈        For    7321            BamHI

NadA ₃₅₀       Rev    7322            XhoI

NadA ₃₃₂       For    7323            HindIII

NadA ₄₀₅       Rev    7324            XhoI

Individual chip is cloned into the pET21b carrier successively, marking protein under derivable T7 promoter control. Use primer S1_L-For and S1_L-Rev has obtained to be fused to the S1 domain (S1 of the spike protein of NadA leader peptide by PCR_L). The forward Oligonucleolide primers contains the restricted sequence of NdeI and the sequence of coding NadA leader peptide and front 6 amino acid of mature protein. The PCR fragment cloned into the NdeI/BamHI fragment in the pET21b carrier of opening with identical Restriction Enzyme. Then with this clone (pET-S1_L) clone successively other different structure territory, such as BamHI/XhoI, BamHI/HindIII or HindIII/XhoI fragment. BamHI and HindIII restriction site can be introduced respectively Amino Acid GS and KL.

The pcr amplification scheme is as follows: with genomic DNA or 10ng DNA goods (the plasmid pCMVnew of 200ng from Neisseria meningitidis 2996, the gene that contains complete coding spike protein) as template, Oligonucleolide primers 40 μ M in the amplified reaction, adopt 400-800 μ M dNTPs solution, 1x PCR buffer solution (contains 1.5mM MgCl₂), 2.5 TaqIDNA of unit polymerases (using Perkin-Elmer AmpliTaQ or Invitrogen Platinum Pfx archaeal dna polymerase).

With entire mixture 95 ℃ cultivate in advance 3 minutes after, every duplicate samples is carried out the amplifications in two steps: carry out front 5 with the hybridization temperature (Tm1) of eliminating primer restriction enzyme tail and take turns. Then, the hybridization temperature (Tm2) that calculates according to full length rna oligonucleotide carries out 30 takes turns. Time 68 ℃ or the 72 ℃ extensions of carrying out is different according to the length of the fragment that will increase. Circulating in 68 ℃ or 72 ℃ 10 minutes extends in the steps and finishes.

The DNA of amplification is directly loaded onto on the Ago-Gel, and by manufacturer's explanation, uses Qiagen^TMGel Extraction Kit is from the dna fragmentation of gel-purified corresponding to correct size strip.

DNA and plasmid vector with the suitable Restriction Enzyme digestion purifying corresponding with amplified fragments carry out purifying and carry out coupled reaction with QIAquickTM PCR purification kit (according to the explanation of manufacturer).

To connect product and be transformed into the competence bacillus coli DH 5 alpha, and by making the clonal growth of selecting at random and extracting DNA by the explanation of manufacturer with Qiagen QIAprep Spin Miniprep kit and screen recombinant clone.

Recombinant plasmid is introduced the e. coli bl21 (DE3) that is used as expressive host. Substance group colony is inoculated in the LB+ ampicillin, cultivated 14-16 hour at 37 ℃. Bacterium can directly be reclaimed by centrifugal (non-inductive condition), or it is diluted to fresh culture medium and 37 ℃ of cultivations, until OD₆₀₀Between 0.4-0.80. Add 1mM isopropylthio-β-D-galactoside (IPTG) induced protein and express 3 hours (inductive condition).

Bacterium is resuspended in SDS-sample buffer 1X and boils obtained full product of cell lysis in 5-10 minute. Explanation according to manufacturer separates equal protein matter with NuPAGE (Invitrogen) or BIORAD gel systems. Protein manifests by Coomassie blue stain or transfers to nitrocellulose filter and carry out the western engram analysis and manifest. The Wester trace NadA of antivenom purification_351-405Rabbit polyclonal antiserum (1: 3000 dilution) and the SA (DAKO) of peroxidase-coupling carry out.

Figure 38 and 39 has shown S1_L、S1 _L-NadA and S1_L-NadA _{The Δ anchor}Result at expression in escherichia coli. Figure 37 has shown fusion construct.

The SARS virus antigen of bacterial expression also can be used to prepare the composition that contains outer membrane vesicles, and wherein said outer membrane vesicles comprises one or more SARS virus antigen.

Outer membrane vesicles (" OMV ") is also referred to as bubble, refers to that adventitia fragment by Gram-negative bacteria forms or by its vesica that is derived. OMV comprises outer membrane protein (OMP), lipid, phosphatide, all metallic substance and lipopolysaccharides (LPS) usually. In the processing that is referred to as alveolation, Gram-negative bacteria forms OMV usually by lytic infection the time. Also can pass through some chemical modification processes, extract such as detergent, obtain OMV from Gram-negative bacteria. Also can prepare lipid bilayer with SARS virus antigen of the present invention and be wrapped in synthetic OMV or a liposome that contains core water.

OMV of the present invention is preferably and contains the lipid vesicle that contains core water that lipid bilayer centers on. This lipid vesicle has single layer structure (namely lipid bilayer centers on contain core water) usually, although multilayer lipid vesicle also can be used for composition of the present invention. The size of OMV usually at nanomole to micro-molar range, for example from about 1nM to 100 μ M, more generally from about 10nM to 10 μ M, preferably from 30nM to 1 μ M.

OMV of the present invention should prepare from Gram-negative bacteria. Gram-negative bacteria is that those can not resist the bacterium that decolouring is processed in the Gram's stain of knowing. Gram-negative bacteria is take the polygon cell membrane of complexity as feature, and usually has outer polysaccharide capsule. The Gram-negative bacteria that is fit to make OMV comprises, for example, neisseria, Moraxella, Kingella genus, acinetobacter, Brucella, the special Pseudomonas of Boulder, chlamydiaceae, fungi, Actinobacillus, Borrelia (Borelia), Serratia, campylobacter, Helicobacterium, Haemophilus spp, Escherichia, Legionnella, Salmonella, pseudomonas and Yersinia.

OMV of the present invention should contain one or more SARS virus antigens or its fragment. SARS virus antigen can be recombinant expressed in the Gram-negative bacteria host cell, then gathers in the crops together with OMV.

The antigenicity component such as recombinant expressed SARS virus antigen, can be arranged in any one or all compartment of three main compartments of lipid vesicle, and these compartments comprise the compartment that invests lipid vesicle inner surface or outer surface by for example film anchoring structure territory; Insert the compartment of lipid bilayer, for example wherein said antigenicity component self is hydrophobicity or based on the entity of lipid; Or be positioned at the compartment of the moisture center of lipid vesicle or core.

OMV or the liposome of synthetic preparation can be used for the present invention. This liposome comprises many different lipids or aliphatic acid. The lipid that is fit to be contained in the liposome of the present invention includes but not limited to phosphatidylinositols-(4,5)-diphosphonic acid, phosphatidylserine, phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidyl glycerol, cholesterol, beta-oil acyl-γ-palmityl, lipopolysaccharides and galactocerebroside (galactocerbroside).

The appropriate method of extracting OMV from the bacterium raw material comprises that dexycholate extracts, Tris/HCl/EDTA extracts and lithium acetate extracts. Preferred described leaching process comprises with physics and/or chemical method and destroys the bacterial cell adventitia so that OMV fully discharges to carry out purification and separation. Referring to for example, WO 03/051379.

Can be by methods known in the art with OMV enrichment of the present invention and/or add therein the antigenicity component, such as SARS virus antigen, these methods comprise, for example, directly in external mixing, wherein can choose wantonly and carry out the high energy mixing so that the antigenicity component is integrated into a compartment of liposome. Be applicable to high energy mixing method of the present invention and comprise homogenize, hypervelocity fragmentation, extruding and their combination.

The preferred antigens component such as SARS virus antigen, is to make with the host cell restructuring that obtains OMV. In one embodiment, this OMV makes by the nucleotide sequence of encoding SARS viral antigen is introduced recombinant host cell. The nucleotide sequence of optimized encoding SARS virus antigen is controlled by the strong promoter sequence. The nucleotide sequence of optimized encoding SARS viral antigen also comprises the adventitia phasing signal. For example, the nucleotide sequence of encoding SARS viral antigen can be fused to the sequence of the outer membrane protein of the natural generation of coding bacterial host. The nucleotide sequence of optimized encoding SARS virus antigen is fused on the signal peptide sequence of outer membrane protein of the natural generation of bacterial host.

The method of making the best OMV that is used for vaccine is described in, for example, and Filip etc., J.Bact. (1973) 115:717-722; Davies etc., J.Immunol.Method (1990) 143:215-225; With WO 01/09350.

In one embodiment, bacterial host cell such as Escherichia coli, is converted to express the SARS spike protein. As mentioned above, spike protein can be beneficial to bacterial expression and spike protein is transported to host cell surface by modification. Above-mentioned various furcella/NadA fusion construct can be used for preparing OMV of the present invention. The construction that preferably contains the furcella S1 ballhead domain that is fused to NadA handle zone is used to produce OMV. This construction can be chosen wantonly and contain NadA leader peptide and NadA anchoring peptide. The structural representation of these preferred OMV constructions is illustrated in Figure 49.

Embodiment 6 has described a kind of method of preparation OMV of the present invention.

(b) mammal of SARS subunit vaccine is expressed

As mentioned above, mammalian host cell is used to recombinant expressed SARS virus albumen. Being applicable to mammalian host cell of the present invention comprises, for example, Chinese hamster ovary (CHO) cell, HeLa cell, baby hamster kidney cell (BHK) cell, MK cells (for example, Hep G2), Madin-Darby ox kidney (" MDBK ") cell and other cell. The mammal source of cell comprises, but be not limited to, the mankind or non-human primates cell are (for example, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), human embryonic kidney cell's (293 cells, usually the adenovirus type 5DNA that is sheared transforms), monkey kidney Vero cell (comprising the COS7 cell), horse, ox (such as the MDBK cell), sheep, dog (dog kidney mdck cell for example, ATCC CCL34MDCK (NBL2) or MDCK 33016 (be DSM ACC 2219 such as deposit number as described in the WO 97/37001), cat and mouse (hamster cell for example, such as BHK21-F, HKCC cell or Chinese hamster ovary cell (Chinese hamster ovary celI)), and can available from each stage of development, comprise for example adult, neonate, fetus or embryo.

The polynucleotides of encoding SARS virus protein can be beneficial to or strengthen expression by modification. For example, the targeting sequencing of this field known available such as tPA or IgK or interleukin 2, can be used for the recombination to construct thing. Yet, preferably use natural SARS targeting sequencing. Use natural targeting sequencing can guarantee that protein will enter the human cell to infect identical approach with normal virus, this dna vaccination of expressing antigen for for example original position is favourable.

As mentioned above, flag sequence can be used for expressing construction so that the protein of purifying, detection and stably express. Be applicable to labelled protein of the present invention comprise poly arginine mark (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin binding peptide, cellulose binding domain, SBP-tag, chitin in conjunction with territory, glutathione s-transferase mark (GST), maltose-binding protein, tanscription termination antitermination factor (transcription termination anti-terminiantion factor) (NusA), escherichia coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). Preferred labelled protein comprises His-tag and GST. Can be referring to Terpe etc. about discussing in detail of labelled protein, " labelled protein merges summary: from molecule and biochemical basis to commercial system ", Appl Microbiol Biotechnol (2003) 60:523-533.

After the purifying, labelled protein can be chosen wantonly from the fusion of expressing and remove, and namely shears enzyme by the known specificity in this field and processes. Normally used protein comprises enterokinase, marmor erodens (TEV), fibrin ferment and X_aThe factor.

One or more amino acid sequences of spike protein or the amino acid structure territory can be removed so that mammal is recombinant expressed. For example, whole S2 domain or furcella cross-film district can be removed. Some of the total length that suitable mammal is expressed or the spike glycoprotein of brachymemma are expressed the representative example of constructions and are seen Figure 40. The polynucleotide sequence that represents each construction is seen SEQ ID NOS 6578-6583. The implication of each symbol is as follows:

Clone's title is described and is expressed construction

The natural targeting sequencing SEQ of nShA ID NO:6578

The total length furcella

Histidine mark

The natural targeting sequencing SEQ of nS ID NO:6579

The total length furcella

The natural targeting sequencing SEQ of nSh Δ TC ID NO:6580

Furcella without the cross-film sequence

Histidine mark

The natural targeting sequencing SEQ of nSh Δ TC ID NO:6581

Furcella without the cross-film sequence

The natural targeting sequencing SEQ of nS1h ID NO:6582

The S1 domain

Histidine mark

The natural targeting sequencing SEQ of nS1 ID NO:6583

The S1 domain

To comprise to form respectively among clone's the cDNA fragment inserting expressioning carrier pCMVIII of furcella construction (TM-Cy-delete furcella) in total length furcella coded sequence and deletion membrane spaning domain and cytoplasmic structure territory and secrete property nSh and nSh Δ TC. These two kinds of spike proteins all contain six histidine marks to help preliminary qualitative expression's spike protein at the C-end. The similar furcella in coding total length furcella or deletion membrane spaning domain and cytoplasmic structure territory but the sequence that do not contain histidine " mark " is that the technical staff who is proficient in this field substitutes easily.

With method shown in Figure 48 with the Separation of Proteins of expressing become to contain water section (AF) and detergent partly (DF) assess may locating of expressed furcella construction, Figure 43 has shown the result of western engram analysis. With their expression that has been transfected into the post-evaluation of COS7 cell of above-mentioned vector construct. The construction of expressing the total length spike protein is retained in cell membrane, and the construction of expressing the spike protein of brachymemma then is arranged in cytosol (Figure 43) or is secreted into cell culture medium (Figure 44). As shown in figure 43, find that the total length spike protein appears at DF (film) with the cohesion form, and the protein of brachymemma appears at AF (cytosol) with monomeric form. As shown in figure 44, (Sh Δ TC) is secreted for the protein of deletion, and has detected a small component of total length spike protein in culture medium with rabbit anteserum.

Recombinant expressed spike protein can oligomerization. When spike protein was used to vaccine or produce the spike protein specific antibody, they are oligomerization preferably. For obtaining the spike protein of oligomerization, preferably membrane spaning domain is retained in the recombinant expression construct thing. For example, Figure 41 compared use anti--his tag and rabbit anti--the Western trace result of the COS7 cell lysate that SARS antibody obtains and the nSh of expression and nSh Δ TC. As shown in the figure, total length (nSh) has formed cohesion, and (nShATC) spike protein of brachymemma then can not. The total length of the Identification of the antibodies native form of the protein of anti-His-mark and reduction form and the spike protein of brachymemma. Rabbit anti-serum is only identified spike protein under non-reduced condition. Furcella agglomerate or oligomer appear in the cell lysate of nSh construction of expression in a large number. The spike protein of preferred described oligomerization forms homotrimer, as shown in figure 47.

Another test, as shown in figure 42, the oligomerization that proves the nSh construction of expressing may be (rather than because, for example, disulfide bond) because non-covalent connection. This oligomer is dissociated into monomer under the temperature (80-100 ℃) that raises, if but not heat under reducing condition be stable.

Recombinant expressed spike protein is also preferably by glycosylation. Useful tunicamycin and glycosidase are assessed glycosylation. Figure 45 explanation, the glycosylation of the spike protein of expression can not removed the impact in membrane spaning domain zone. (Sh Δ TC) SARS spike protein of total length (Sh) and brachymemma is all by glycosylation.

Preferred expression construction of the present invention is to the mammalian host cell avirulence. Figure 46 proves that the construction of furcella shown in the expression is to COS7 host cell avirulence.

The method of recombinant protein is known in the art in transfection, expression, cultivation, separation and the purifying mammalian cell cultures. For example, SARS furcella construction of the present invention can be at 293 cells. These cells can be cultivated and transfection in static culture or monolayer cultivation thing. The SARS proteantigen of making capacity for rapid large-scale comprises immunogenicity research to carry out external or interior evaluating, can extensive of short duration transfection 293 (human embryo kidney (HEK)) cell to obtain the recombinant antigen of milligram order of magnitude. Perhaps, can be in suspension culture extensive rotaring redyeing 293 cell. The SARS albumen of preferred expression be 48-72 after the transfection hour or even transfection after 72-96 hour or more time from the cell harvesting of transfection.

When expressing the construction transfection host cell with the furcella of brachymemma, the spike protein of expression is collected by secretory host cell and from cell culture medium. For example can adopt after concentrated the GNA agglutinin then by DEAE and ceramic hydroxyapatite column chromatography from culture medium purifying spike protein.

When expressing the construction transfection host cell with the total length furcella, the spike protein of expression still is retained in the cell, can be from the cell purification that extracts with triton X-100 detergent. Then available GNA agglutinin is caught the total length spike protein, again by hydroxyapatite and SP chromatographic purifying.

Also can produce Chinese hamster ovary (CHO) or other eucaryon (for example, mammal) cell (for example Figure 73) of stably express SARS virus antigen of the present invention. Preferred described cell is Chinese hamster ovary celI, and these constructions will contain one or more labels or Select gene to select required Chinese hamster ovary celI. In one embodiment, for the screening purpose, described construction comprises DHFR and the attenuation neomycin gene of cmv enhancer/promoter, ampicillin resistance gene and fusion. Then available neomycin selective system produces stable clone in the CHOK-1 cell. Then the clone of screening can be sequenced to verify the integrality of identifying insert, then available Trans-LT1 polyamine transfection reagent (PanVera Corp., Madison, WI) carry out of short duration transfection with the evaluation expression level, and the integrality by ELISA and western engram analysis evaluation expression protein.

The method of the Chinese hamster ovary celI of the stably express of deriving SARS virus antigen of the present invention comprises the transfection step and with the step of selective medium Preliminary screening. Randomly, can carry out subclone to guarantee the purity of clone after these steps. Available antigen capture ELISA measures cell culture supernatant with quantitative selection and the expression in all stages of increasing.

For the total length furcella was expressed construction, the anti-SARS antibody of available rabbit carried out the intracellular expression of immunofluorescence dyeing screening methyl alcohol fixed cell. Carry out continuous measurement to guarantee the stability of expression in the T75 flask amplification stage. Can then survey to verify molecular weight and the integrality of marking protein by immunity by PAGE (natural endowment and the condition that reduces denaturation).

In one embodiment, will express the pCMV3 carrier of total length or clipped form SARS-CoV spike protein with Trans-LT-1 reagent and introduce the CHOK-1 cell. The 1st day, with 1 * 10⁶Cell is seeded on the 100mm plate, contains non-selective F12 culture medium+10% hyclone+4mM glutamine in the plate. The 2nd day, use DNA:LT-1 mixture transfectional cell, and replace original culture medium with complete F12 culture medium. According to cell density, after 24-48 hour, each 100mm plate is divided into 4-6 100mm plate. Culture medium is changed into the complete selective medium that contains 500 μ g/ml Geneticins (neomycin). The cow's serum of these methods that are useful on meets the current standard of FDA all without the TSE source. After 24 hours culture medium is changed into the complete selective medium of interpolation 500 μ g/ml neomycins. After 10-14 days, select single bacterium colony, it is transferred to 96 orifice plates and in not containing the complete selective medium of G418, cultivate. When the hole when about 80% was paved with, 24 hours supernatants of furcella being caught the selection of ELISA positive colony were transferred to 24 orifice plates. Express for starting the total length spike protein, with rabbit anti--SARS antibody makes the fixing cell of immunofluorescence dyeing screening methyl alcohol. Also have less than 20-30 clone after rejecting low expression clone, measure expression with catching ELISA and western after the lysis. Precipitate each clone a part, weigh, (contain MOPS, NaCl and MgCl with the 1%triton lysis buffer of cell and lysis buffer identical weight ratio₂) cracking. Collect supernatant after the cracking and measure expression. 3-4 is produced being cloned in 3 liters the bioreactor of the correct spike protein of structure and conformation with highest level cultivate amplification and adapt to low serum condition of suspension culture, with the expansion scale.

Can carry out antigen capture ELISA to the SARS spike protein by the description in this field measures. To being summarized as follows of this assay method. The immunoglobulin (Ig) available from the rabbit anteserum of using the deactivation of SARS virus immunity of coating 250ng purifying in each hole of the flat flat boards in 96 holes (Corning, Corning, NY). Then dull and stereotyped with the buffer solution washing that contains 16%NaCl and 1% Triton X100. Adding 100 μ L supernatants or lysate sample (is diluted in and contains 100mM NaPO₄, 0.1% casein, 1mM EDTA, 1%Triton X100,0.5M NaCl and 0.01% thimerosal, in the buffer solution of pH 7.5) and cultivated 2 hours at 37 ℃. In conjunction with antigen and the SARS+ve serum of collection or neutralizing high affinity human or mouse-anti SARS spike protein monoclonal antibody reactive (cultivating 1 hour for 37 ℃), and detect (37 ℃ of cultivations 30 minutes with suitable species specificity peroxidase conjugated SA; TAGO, Burlingame, CA). Flat board is at room temperature used TMB (Pierce, Rockford, IL) colour developing and with 4N phosphoric acid cessation reaction. Read flat board at 450nm, produce the concentration of protein in every ml sample according to the calibration curve (OD is to protein concentration) of concentration known restructuring spike protein serial dilution making.

After the standard method that this area has been described, also can carry out immune detection analysis. Be summarized as follows. The 4-20%SDS PAGE of mild heat analyzes 10-20 μ l sample under non-reduced/Denaturing. Under the 100V constant voltage, run 1.5-2.0 hour gel. Then according to the explanation of manufacturer, use half-dried western transfer system (BioRad, Hercules, CA) that protein transduction is moved on to nitrocellulose filter (Millipore, Bedford, MA), last 45 minutes. Make the anti-furcella seroreaction of film and multi-clone rabbit, again with put together in Alexa 688 anti--rabbit Ig (Molecular Probes, Oregon) reaction. With infrared imaging system (LI-Cor, Inc., Lincoln, Nebraska) scanning trace.

Filter out spike protein expression candidate cell the highest and that stability is the highest in small-scale (3 liters) suspension culture system. Further estimate the expression of this candidate clone and the integrality of marking protein after the amplification, and the expression stability when detecting subsequently without candidate. Can also monitor selected clone and keep the ability of complete S ARS spike protein gene DNA sequence integrality. Be the little flask of Fast Monitoring (T25 or T75) and 3 liters of expressions of estimating in the culture, available method based on agglutinin (Gluvanthus Nivalis agglutinin) is separated into the SARS spike protein can carry out sxemiquantitative and purity qualitatively to the protein in the CHO supernatant. For the total length spike protein, can obtain from the cell that triton X-100 detergent extracts. Then the total length spike protein is captured on the GNA agglutinin, then carries out hydroxyapatite and SP chromatography. The protein of qualitative wash-out by the following method: 1) polyacrylamide gel electrophoresis (PAGE) and coomassie dyeing, 2) resist-detection of SARS sero-immunity with rabbit, 3) carry out structural analysis with size exclusion chromatography (SEC), and carry out mass spectral analysis with MALDI-TOF.

The approaches and methods that carries out immunity with vaccine of the present invention will be described in detail in following part. Embodiment 7-9 has exemplified the scheme of restructuring spike protein immunity inoculation.

The vaccine test

Be used for before the human body, usually need to be in animal model test vaccine. The mouse model (Subbarao etc. (2004) J Virol 78:3572-77) that known sars coronavirus infects, other animal that can be used as infection and/or disease model comprises ferret and monkey. Therefore, the invention provides the non-human animal that sars coronavirus infects, wherein, the preferred ferret of described animal or primate (for example monkey or macaque). Animal can be germfree animal. Preferred animal is not cat (Felis domesticus). This animal can show or not show the SARS disease symptoms, and (Mustela furo) demonstrates obvious lung pathology after infection such as ferret. Referring to: Martina etc. (2003) Nature 425:915.

E. code book is invented the polynucleotides of SARS antigen

The present invention includes the polynucleotides of code book invention SARS antigen. In addition, the present invention includes optimization (as by the codon optimization) restructuring and make the polynucleotides of SARS antigen, comprise the polynucleotides of above-mentioned each SARS fusion construct of encoding.

F. carry viral vectors or the virion of SARS antigen of the present invention

Antigen of the present invention can be by the polynucleotides of coding for antigens in vivo or vivoexpression. Help expression and the conveying of polynucleotides of the present invention by viral vectors and/or virion.

Give heterologous gene in the external and body of the gene delivery system that available virus (such as α virus) produces, comprise that one or more have the SRAS gene for the treatment of or preventive use. Also available these systems make in the cell of cultivating and are derived from the SARS virus recombinant protein. Induction system based on gene of the present invention comprises the non-viral nucleic acid carrier (for example, DNA, RNA) of viral vectors (for example, adenovirus vector, poxvirus vector, α viral vectors) and the one or more SARS virus antigens of coding. (for example as the bicistronic mRNA construction) is incorporated in this gene vaccine the polynucleotides of encoding SARS viral antigen by alone or in combination.

1. α is viral

α virus is that the member of Togaviridae has common structure and duplication characteristic. Sindbis virus (SIN) is the prototype virus that other α virus is carried out molecular studies, is α virus (Schlesinger and Dubensky (1999) the Curr Opin.Biotechnol.10:434-439 that can produce the allogeneic gene expression carrier of the most extensive employing with Venezuelan equine encephalitis virus (VEE) and Semliki forest virus (SFV); Schlesinger (2001) Expert Opin.Biol.Ther.1:177-91).

α virus has the normal polarity single stranded RNA genome of less, and its length is about 12kb, is added cap and is gathered adenosine. RNA and viral capsid proteins monomer interact and form nucleocapsid, and then nucleocapsid is centered on by the lipid envelope that host cell is derived again, and two VGP E1 and E2 just can form heterogeneous dimer subunit " furcella " tripolymer from wherein protruding. The polyprotein of two ORFs (ORF) coding is arranged, and is unstructuredness replicase protein matter (5 ' ORF) and the virus particle structure protein (3 ' ORF) with enzymatic activity. Structural polyprotein is from highly abundant subgenomic mRNA translation, and described subgenomic mRNA is (Strauss and Strauss (1994) Microbiol.Rev.58:491-562) of the potent promoter transcription of α virus internally. This genomic copying as RNA only occurs in the kytoplasm of host cell.

The most frequently used α virus expression carrier utilizes normal chain characteristic and the modular structure of rna gene group. The characteristic that has spontaneous amplification owing to these carriers is called as " replicon ", they allow heterologous sequence to insert and substitute this structural polyprotein gene, keep simultaneously 5 '-and 3 '-terminal cis reproducing signals, unstructuredness rdrp gene and sub-gene group land promoter (Xiong etc., (1989) Science 243:1188-1191; Liljestrom (1991) Bio/Technology 9:1356-1361). The mosaic α viral vectors (and particle) that divergence virus family sequence is derived also is described. (referring to, for example, U.S. Patent Application Serial 09/236,140; Also can be referring to United States Patent (USP) 5,789,245,5,842,723,5,789,245,5,842,723 and 6,015,694; And WO 95/07994, WO 97/38087 and WO 99/18226). Submitting on December 12nd, 2002 and being included in full in this paper is the improved α viruses molecule that total international publication WO 02/099035 has described the Chimeric alpha viruses molecule and had improved biological safety level as a reference.

Lack structural protein gene and make the sub-carrier defective of α virus replication, thereby the expression of RNA amplification and high-level heterologous gene occurs in the target cell, thereby but owing to can't form the propagation that the cell-cell of this carrier can not occur the progeny virus particle. In recent years, some occur and be used for describing the identical term of implication of the sub-particle of α virus replication. These terms comprise recombinant virus particle, recombinant alpha virion, the sub-particle of α virus replication and replicon particle. Yet these terms all represent to contain the Virosome-like unit of the RNA carrier replicon that α virus derives here. In addition, these terms can represent carrier, vector construct or gene delivery carrier.

By cell that replicon rna introduce is allowed (for example, RNA or DNA transfection, or particle transfection) replicon rna can be packaged into particle, contain structural proteins expression cassette or " defective auxiliary " construction of one or more coding for alpha virus structural proteins in the cell of described permission. The structural proteins self of these coding constructions can be introduced into cell by transfection RNA or DNA, and usually keep natural α viral subgenomic because of the group promoter, and 5 '-and 3 '-terminal cis signal with the replicon coamplification, but lack any rdrp gene and RNA packaging signal (Liljestrom (1991) Bio/Technology 9:1356-1361; Pushko etc., (1997) Virology 239:389-401; Polo etc., (1999) PNAS 96:4598-4603). Immortal cell line (for example, package cell line) with the construction stable conversion of express alpha virus structural protein provides a kind of method of avoiding of short duration transfection to produce (Polo etc., (1999) PNAS 96:4598-4603).

(for example the present invention includes to make the replication defective vector particles, the sub-particle of α virus replication) composition and method, to be used for the using heterologous gene that coding has the protein for the treatment of or preventive use in external and the body, comprise the gene of one or more SARS virus antigens of encoding.

On the one hand, the present invention includes and (for example make the replication defective vector particles, the sub-particle of α virus replication) method, the method be included in can with viral vectors (for example, the sub-RNA of α virus replication) complementation and manufacturing vector particles are (for example, the sub-particle of α virus replication) (for example contains viral vectors with at least a under the condition, the sub-RNA of α virus replication) nucleic acid molecules is introduced infinite multiplication cell of the present invention, and from the step of cell or cell culture supernatant isolated viral carrier granular. In some embodiments, described unlimited in Growth of Cells in suspension, PERC.6 cell for example. In other embodiments, the method is carried out under larger volume, for example with volume or the more volume of the unit of being upgraded to, as in shaking flask, in the large flask, Nunc Cell Factories, Corning Cell Cubes, fermentation tank be medium).

In some embodiments, described viral vectors is to provide one or more trans α virus structural proteins to make α virus replication that replenishes in the infinite multiplication cell. At this moment, be included in by the method that replenish to produce the sub-particle of α virus replication and introduce one or more in the infinite multiplication cell and (for example encode described α virus structural protein, capsid and/or envelope glycoprotein) nucleic acid (for example, RNA, DNA), this can be temporary transient or permanent, and can carry out simultaneously or carry out before with the sub-RNA of introducing α virus replication. In some embodiments, the RNA by transfection in-vitro transcription replicon rna introduces cell with the sub-RNA of α virus replication. The DNA that can transcribe in cell by transfection in other embodiments, (for example, ELVIS) introduces cell with the sub-RNA of α virus replication. Again in another embodiment, by dying with the sub-particle kind of α virus replication liquid inductance the RNA of α virus replication is introduced cell. In some embodiments, the nucleic acid of the described virus structural protein of encoding is the complementary RNA of defective or the DNA that can transcribe the complementary RNA of defective in cell.

" α viral RNA replicon carrier ", " the sub-carrier of rna replicon ", " replicon carrier " or " replicon " refer to guide the RNA molecule of self-replication in himself amplification or the body here in target cell. For guiding the amplification of himself, this RNA molecule should encode the essential polymerase of catalysis RNA amplification (for example, α virus non-structural protein nsP1, nsP2, nsP3, nsP4) and contain simultaneously the polymerase that is encoded identify and utilize copy required cis RNA sequence. α viral rna vector replicon should contain the element of following order: required 5 ' virus sequence or the cell sequence of non-structural protein mediation amplification (is also referred to as 5 ' CSE, or 5 ' cis replication sequence, or copy required cis 5 ' virus sequence, maybe can start 5 ' sequence of α virus transcription), encoding human active alpha virus non-structural protein (for example after expression, nsP1, nsP2, nsP3, nsP4) sequence, and required 3 ' virus sequence or the cell sequence of non-structural protein amplification (is also referred to as 3 ' CSE, or copy required cis 3 ' virus sequence, or the α viral rna polymerase is identified sequence). α viral rna vector replicon also comprises a kind of component and expresses one or more heterologous sequences, such as IRES or virus (for example, α virus) sub-gene group promoter (for example, the land promoter), in some embodiments, this promoter can be modified to strengthen or be reduced virus transcription sub-gene group fragment, or the homology of reduction and the auxiliary auxiliary of defective or structural proteins expression cassette and one or more heterologous sequences to be expressed. Described heterologous sequence preferably comprises the gene of coded protein, and it is near the 3 ' gene of holding in the carrier replicon. Described replicon also comprises the polyadenylic acid sequence.

" recombinant alpha virion ", " the sub-particle of α virus replication " and " replicon particle " represent to contain the Virosome-like unit of α viral rna vector replicon here. Usually, described recombinant alpha virion comprises one or more α virus structural proteins, lipid envelope and RNA carrier replicon. Preferred described recombinant alpha virion comprises the nucleocapsid structure, and this structure is contained in the lipid bilayer that host cell derives, and such as cytoplasmic membrane, wherein is embedded with one or more α viral envelope glycoproteins (for example, E2, E1). This particle also can contain other component (for example, target element such as biotin, other virus structural protein or its part, hybridization coating or other receptors bind part), and these components can guide the preferendum of the α virion of deriving. Usually, effectively forming interaction between the necessary α viral RNA of α replicon particle or nucleocapsid and the structural proteins may be the RNA-protein interaction between contained packaging signal or packaging sequence in capsid protein and the RNA.

The cell that " α Viral packaging cell system " refers to contain one or more α virus structural protein expression cassettes and make the recombinant alpha virion after introducing α viral rna vector replicon, eucaryon layered support start-up system or recombinant alpha virion. Parental cell can be from mammal or nonmammalian. In preferred embodiments, described package cell line is by structural proteins expression cassette stable conversion.

" the complementary RNA of defective " refers to can be at the RNA molecule of one or more α virus structural proteins of eukaryotic expression that also contain functional alpha virus non-structure " replicase " albumen through amplification. Described α virus non-structural protein can be by α viral RNA replicon carrier or other means at cell inner expression. For increasing and expression structure albumen (by the mediation of α virus non-structural protein), the complementary RNA molecule of described defective should contain 5 ' required-terminal and the 3 '-terminal RNA sequence of amplification that can be identified by non-structural protein and utilize, and the component of expressing one or more α virus structural proteins. Therefore, the complementary RNA of α virus defective (is also referred to as 5 ' CSE because of required 5 ' virus sequence or the cell sequence of RNA amplification that contains the element of following order: α virus non-structural protein, or 5 ' cis replication sequence, or copy required cis 5 ' virus sequence, maybe can start 5 ' sequence of α virus transcription), express the component of one or more α virus structural proteins, after expression, encode one or more α virus structural proteins (for example, C, E2, E1) gene order, required 3 ' virus sequence or the cell sequence of α virus non-structural protein amplification (is also referred to as 3 ' CSE, or copy required cis 3 ' virus sequence, or the α viral rna polymerase is identified sequence), and preferably contain the polyadenylic acid sequence. Usually, the complementary RNA of described defective self does not encode or all four kinds of α virus non-structural proteins (nsP1, nsP2, nsP3, nsP4) of expressed intact, but codified or express subclass or its part of these protein, or contain sequence from one or more nonstructural protein genes, but these sequences that are contained in the defective auxiliary are not expressed non-structural protein or its part. Means as a kind of express alpha virus structural protein, the complementary RNA of described defective (for example can contain virus, α virus) sub-gene group promoter, in some embodiments, this promoter can be modified to regulate transcribing of sub-gene group fragment, or the homology of reduction and replicon rna, or comprise other component (for example, internal ribosome entry site, ribosomes are readed over element) that affects the expression of α virus structural protein. Preferred α virus structural protein gene is near the 3 ' gene of holding in the defective auxiliary. In addition, the also preferred complementary RNA of this defective does not contain the sequence that can promote RNA-protein and α virus structural protein to interact and be packaged into nucleocapsid, Virosome-like particle or the sub-particle of α virus replication. The complementary RNA of defective is a particular embodiments of α virus structural protein expression cassette.

Be used for the clone that α virus of the present invention can be grown in above-mentioned any one suitable SARS virus.

Can according to method of the present invention, make the sub-particle of α virus replication by using above-mentioned clone (for example immortal cell line) and many open and received α viral vectors methods. This method comprises that for example, of short duration packing is sent out, for example the complementary RNA of the replicon of cotransfection vitro conversion and defective (Liljestrom, Bio/Technology 9:1356-1361,1991; Bredenbeek etc., J.Virol.67:6439-6446,1993; Frolov etc., J.Virol.71:2819-2829,1997; Pushko etc., Virology 239:389-401,1997; United States Patent (USP) 5,789,245 and 5,842,723), or the cotransfection DNA copies son and the complementary construction of defective (Dubensky etc., J.Virol.70:508-519,1996), and with α virus structural protein expression cassette (for example, DNA defective auxiliary) introduces stable package cell line (PCL) (Polo etc., PNAS 96:4598-4603,1999 of generation in the immortal cell line of the present invention; United States Patent (USP) 5,789,245,5,842,723,6,015,694; WO 97/38087, and WO 99/18226, and WO 00/61772, and WO 00/39318). Then stable package cell line can be used for making the sub-particle of α virus replication. PCL can use the sub-RNA transfection of α virus replication of in-vitro transcription, (for example copy son with DNA, the ELVIS carrier) transfection, or with the transfection of the sub-particle kind of α virus replication liquid, then cultivate under certain condition the enough time of in the culture supernatant, making the sub-particle of filial generation α virus replication. In addition, subsequently can be by infecting the filial generation replicon particle that in other natural PCL culture, goes down to posterity, this will cause further increasing and obtaining commercial-scale goods. Importantly, can detect the replicon particle storage liquid that pollutes RCV by using the complementary RNA of defective or the stable PCL based on " breach " structural gene configuration, can generate not contain.

After the results, can make cutting pass through filter membrane (for example, aperture 0.2uM, 0.45uM, 0.65uM, 0.8uM) and clarify the rough culture supernatant that contains the sub-particle of Chimeric alpha virus replication. Before filtering, can choose wantonly rough supernatant is carried out low-speed centrifugal to remove than the maxicell fragment. In one embodiment, in the sub-particle product of α virus replication, adding endonuclease (for example, Benzonase, Sigma#E8263) before or after the chromatographic purifying step with the digestion exogenous nucleic acid. In addition, available any method (for example, tangent flow ultrafiltration) known to widely concentrates these goods before purifying. Can be by chromatographic technique (for example, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography) come concentrated and purification of crude or the sub-particle of α virus replication through clarifying, as described in WO01/92552, this application is included in this paper as a reference in full. Can use successively two or more these type of purification process.

The example of the sub-particle of α virus replication of encoding SARS viral spikes antigen

The present invention includes and (for example make the replication defective vector particles, the sub-particle of α virus replication) composition and method, the protein that described carrier granular can be used for encoding in external and the body has the heterologous gene for the treatment of or preventive use, comprises the gene of one or more SARS virus antigens of encoding.

Following examples have exemplified the method for the sub-particle of α virus replication of preparation encoding SARS virus furcella antigen.

SARS virus furcella gene can be impregnated in the sub-particle of α virus replication from various α virus (such as sindbis virus, Semliki forest virus (US 5739026), Venezuelan equine encephalitis virus (US 6531135) and from the replicon particle chimera (US 6376236, and WO 02/99035) of more than one α viruses). In addition, SARS virus furcella gene can its complete form (fgs encoder total length spike protein) or is mixed with modified forms, described modified forms comprises, for example, sequence deletion or brachymemma, so the length of the spike protein of coding has been deleted cross-film district or kytoplasm tail less than total length (for example, that the C-end is clipped is one or more (such as at least 1,2,3,4,5,6,7,8,9,10,15,20,25,30 etc.) amino acid).

For example, can be by standard RT-PCR conditioned disjunction by the standard subclone, from one of other plasmid described here, use commercially available restriction endonuclease, the furcella gene be can be used as full-length gene be cloned into VCR-chim2.1 carrier (WO 02/99035). For carrying out the reverse transcription step among the RT-PCR, used the Superscript kit (Invitrogen that increases in advance^TM) and primer SEQ ID NO:7325 (sp-RT-R):

Amplification step is used cDNA polymerase advantage kit (Clonetech) and two primer Sp-F-BbvCI (SEQ ID NO:7326) and Sp-R-NotI (SEQ ID NO:7327):

The forward primer of design contained sequence ccacc (Kozak, 1991 JBC 19867-70) before the ATG codon so that the translation efficiency of furcella gene is optimum. Simultaneously, forward primer contains the BbvCI restriction site, and reverse primer contains the NotI restriction site so that the gene of subsequently clone PCR amplification.

PCR product QIAquick Nucleotide Removal kit (QIAgen) purifying, with BbvCI and NotI digestion, with QIAquick Gel Extraction kit (QIAgen) gel-purified, and be connected to on the predigested plasmid VCR-Chim2.1 of identical enzyme. The clone who contains SARS furcella sequence checks by checking order, and new construction is called as VCR-Chim2.1-SARSspike.

For producing VEErep/SINenv-SARSspike replicon particle, with Restriction Enzyme PmeI with plasmid VCR-Chim2.1-SARSspike, VCR-DH-Scap (WO 02/99035) and VCR-DH-Sglydl160 (WO 02/99035) linearisation, and by top description (Polo etc., 1999, PNAS 96:4598-603; WO02/99035) carry out in-vitro transcription. And by top description (Polo etc., 1999, the same; WO02/99035) the transcript cotransfection is entered bhk cell. The cell of transfection is collected supernatant after 20-30 hour 34 ℃ of cultivations behind the electroporation, by centrifugal clarification, and by chromatographic purifying, as mentioned above (WO 01/92552).

Infecting the spend the night SARS spike protein of the sub-particulate vector of checking copying of bhk cell with the VEErep/SINenv-SARSspike of purifying or VEErep/SINenv-GFP (WO 02/99035) replicon particle expresses. In addition, bhk cell is also used the parallel transfection of VCR-Chim2.1-SARSspike replicon rna of in-vitro transcription. Infect rear 16 hours cracking infection cells, and pass through western engram analysis lysate sample with the antibody that can identify the SARS virus spike protein. Protein on the gel is colored or transfers on the film to carry out the Western engram analysis, analyzes the serum that adopts rehabilitation or mouse or the rabbit anti-serum of anti-SARS virus and carries out. Such as the description of other parts of the present invention, give vaccine acceptor (for example, rodent, non-human primates, the mankind) with VEErep/SINenv-SARSspike replicon particle.

Figure 67 has shown the western engram analysis result's that use SARS virus specificity rabbit polyclonal antiserum obtains under non-reduced condition data. Western data acknowledgement, SARS spike protein are not only at the cell inner expression that is infected or be replicated sub-RNA transfection by the sub-particle of α virus replication, and the advantage form of spike protein is homotrimer (Figure 67 A). The homotype trimerization of having observed similar spike protein the western trace of the SARS virus particle of the VERO cell conditioned medium purifying that infects from SARS virus connects, this homotrimer is heat-labile, and it is dissociated into monomeric form at 80 ℃ and 100 ℃ this point (Figure 67 B) has been described.

For the expression that further characterizes the SARS spike protein and the processing behind the sub-vector expression of α virus replication, infect the BHK-21 cell with the sub-particle of the α virus replication of expressing the total length furcella. After infecting 6 hours with 5MOI, the cell L-[of infection³⁵S] methionine/cysteine mark 1 hour. [³⁵S]-spike protein of mark digests with the anti-SARS sero-immunity precipitation of rabbit and with Endo-H. Digestion and indigested protein all use 4% polyacrylamide-SDS PAGE to analyze under reducing condition. Shown in Figure 55, synthesized the total length spike protein, it is Endo-H responsive type high mannose glycoprotein (gp170, ER form), is modified to the Endo-H resistance glycoprotein (gp180, golgiosome form) with complex oligosaccharide. Gp170 changes the gp180 form into to be finished in 2 hours.

With the sub-particle of α virus replication of expressing one or more SARS albumen (for example, VEErep/SINenv-SARSspike replicon particle) gives the vaccine acceptor (for example to induce the SARS specific immune response, rodent, ferret, non-human primates, the mankind), as described in other parts of the present invention. Immunity inoculation can be undertaken by number of ways, comprising, for example, muscle is interior, subcutaneous, in the corium and in the nose. In addition, the sub-particle of described α virus replication can use separately or unite use (for example " initial immunity-reinforced immunological (prime-boost) ") with other immunization method of the present invention, perhaps, the sub-particle of described α virus replication can coexpression from the antigen of other respiratory disease substance, or use altogether with the sub-particle of α virus replication of expressing from the antigen of other respiratory disease substance (for example, influenza virus, parainfluenza virus, Respiratory Syncytial Virus(RSV), the super Pneumovirinae of people). For example, in mouse, confirmed to have induced anti--spike protein antibody (Figure 68) with VEErep/SINenv-SARSspike replicon particle immune animal. These researchs that mouse is carried out also comprise other vaccine group of comparison, the spike protein vaccine that comprises the described SARS inactivation of viruses of this paper other parts and restructuring brachymemma, and the DNA that is used as primer, then make booster immunization with the sub-particle of α virus replication. Data are clear to show that all vaccine group have very strong immune response, comprising the sub-groups of grains of α virus replication. Should be noted that the antibody horizontal that the used SARS inactivated virus vaccine of these tests is induced shows it is protectiveness in SARS virus animal challenge model.

Similarly, according to method of the present invention and adopt standard molecular biological technique, the gene of other SARS virus antigen (for example, nucleocapsid protein, membrane glycoprotein) of will encoding is cloned into the sub-carrier of α virus replication alone or in combination to produce the sub-particle of α virus replication.

Express the example of SARS virus furcella α virus particle

The present invention includes manufacturing and express the preventative or therapeutic immunization that the DNA of SARS virus antigen infects to carry out anti-SARS virus. In one embodiment, described SARS virus antigen is furcella (S) albumen. In one embodiment, described DNA is based on α virus.

Following examples have exemplified the method based on the DNA of α virus that SARS virus furcella (S) is expressed in a kind of manufacturing.

SARS furcella gene can copy son with any DNA based on α virus and carry, such as ELVS (Dubensky etc., 1996J Virol.70:508-19), SINCP (WO 01/81609) or VCP (PCT WO 02/99035).

For example, with standard RT-PCR technology SARS furcella Gene cloning is entered SINCP. Reverse transcription step adopts oligomerization Sp-RT-R and the Superscript kit (Invitrogen) that increases in advance. Amplification step adopts cDNA polymerase advantage kit (Clonetech) and primer Sp-R-NotI and Sp-F-XhoI (SEQ ID NO:7328).

The Sp-F-XhoI primer of design contained sequence ccacc (Kozak 1991, and is the same) before the ATG codon so that the translation efficiency of furcella gene is optimum. Simultaneously, this primer contains the XhoI restriction site so that the gene of subsequently clone PCR amplification.

PCR product QIAquick Nucleotide Removal kit (QIAgen) purifying, with XhoI and NotI digestion, with QIAquick Gel Extraction kit (QIAgen) gel-purified, and be connected to on the predigested plasmid SINCP of identical enzyme. The clone who contains SARS furcella sequence checks by checking order, and new construction is called as SINCP-SARSspike.

In low blood serum medium Optimem (Life Technologies), add 5 μ l TransIT polyamine reagent (Mirrus) and cultivate in advance the expression of 5 minutes of short duration transfection bhk cell checking SARS furcella genes with 2 μ g DNA SINCP-SARSspike or SINCP. 48 hours cell lysis and the lysis matter sample carried out 8% SDS-PAGE after the transfection. Protein on the gel is colored or transfers on the film to carry out the Western engram analysis, analyzes the serum that adopts rehabilitation or mouse or the rabbit anti-serum of anti-SARS virus and carries out.

(for example give the vaccine acceptor with the SINCP-SARSspike plasmid replicon as the plasmid vaccine through preparation or un-formulated, rodent, non-human primates, the mankind), can use separately or unite use (for example " initial immunity-reinforced immunological ") with other vaccine of the present invention, as described in other place of this paper.

Similarly, the encode gene of other SARS virus antigen (for example, nucleocapsid protein, membrane glycoprotein) is cloned into α virus particle replicon carrier.

2. plasmid expression vector

Express the example of the DNA of SARS virus furcella (S)

Following examples have exemplified the method for making the DNA of expressing SARS virus furcella (S).

SARS virus furcella antigen also available other expression of plasmid DNA carrier (sometimes being called as " routine " dna vaccination) is carried, and this expression vector is based on the polymerase II promoter, such as the CMV promoter. The dna vaccination of furcella antigen gene is induced antibody response (Zhao etc. in mouse; (2004) Acta Biochim et Biophysica Sinica 36:37-41); and be found in mouse, to induce virus to neutralize and protective immunity (Yang etc.; (2004) Nature 428:561-564), especially when C-end during by brachymemma.

For example, Application standard RT-PCR technology enters pCMVKm2 (Zur Megede etc., J.Virol., 74:2628-2635,2000 with SARS furcella Gene cloning; SEQ ID NO:9923). The reverse transcription step is used oligomerization Sp-RT-R and the Superscript kit (Invitrogen) that increases in advance. Amplification step is used cDNA polymerase advantage kit (Clonetech) and primer Sp-F-EcoRI (SEQ ID NO:7329) and Sp-R-XbaI (SEQ ID NO:7330).

The forward primer of design contained sequence C CACC (Kozak, 1991, the same) before the ATG codon so that the translation efficiency of furcella gene is optimum. Simultaneously, forward primer contains the EcoRI restriction site, and reverse primer contains the XbaI restriction site so that the gene of subsequently clone PCR amplification.

PCR product QIAquick Nucleotide Removal kit (QIAgen) purifying, with XhoI and NotI digestion, with QIAquick Gel Extraction kit (QIAgen) gel-purified, and be connected to on the predigested plasmid pCMVKm2 of identical enzyme. The particle that contains SARS furcella sequence is checked by checking order, and new construction is called as pCMVKm2-SARSspike.

In low blood serum medium Optimem (Life Technologies), add 5 μ l TransIT polyamine reagent (Mirrus) and verify the expression of SARS furcella gene with 5 minutes of short duration transfection BHK of 2 μ g DNA pCMVKm2-SARSspike or the pre-cultivation of pCMVKm2 or 293 cells. 48 hours cell lysis and the lysis matter sample carried out 8%SDS-PAGE after the transfection. Protein on the gel is colored or transfers on the film to carry out the Western engram analysis, analyzes the serum that adopts rehabilitation or mouse or the rabbit anti-serum of anti-SARS virus and carries out.

Give vaccine acceptor (for example, rodent, non-human primates, the mankind) with plasmid pCMVKm2-SARSspike as the plasmid vaccine through preparation or un-formulated, as described in other place of this paper.

Similarly, the encode gene of other SARS virus antigen (for example, nucleocapsid protein, membrane glycoprotein) is cloned into plasmid expression vector.

3. the virus-like particle that contains SARS antigen

SARS virus antigen of the present invention can be formulated into virus-like particle (" VLP "). Therefore, the present invention includes the virus-like particle (or VLP) that contains one or more SARS virus antigens. Preferred described VLP comprises that one or more are selected from lower group SARS virus antigen: furcella (S), nucleocapsid (N), film (M) and coating (E). Preferred VLP comprises M and E at least.

VLP of the present invention comprises the polypeptide of at least a formation particle. The polypeptide of described formation particle should be selected from the coronavirus structural proteins. In one embodiment, the polypeptide of described formation particle is selected from one or more SARS virus antigen. In another embodiment, the polypeptide of described formation particle is selected from the structural proteins of non-sars coronavirus (for example MHV).

When expressing, virus structural protein can form VLP in eucaryon or prokaryotic expression system. By expressing, the structural proteins self-assembly forms particle. Perhaps, virus structural protein can be prepared from the intact virus separation and with phosphatide. This virus structural protein is called as " polypeptide that forms particle " here. Owing to there is not viral genome, the VLP right and wrong are infective, yet these non-viral capsids that copy can be simulated the structure of natural viral particle.

Because their structure, VLP can be at the many antigen sites of its surface display (being similar to natural viral). VLP is useful to live vaccine or attenuated vaccine, because they do not have infectivity, their production and use are all very safe. Known VLP can induce antibody neutralization and t cell response, and can present by I class and II class MHC approach.

The previous work that creates coronavirus VLP shows that E and M albumen just are enough to separately form coronavirus VLP. Referring to Fischer etc., J.Virol. (1998) 72:7885-7894 and Vennema etc., EMBO J. (1996) 15:2020-2028.

The present invention also comprises and containing from the polypeptide of the formation particle of non-sars coronavirus or the chimeric VLP of its part. The polypeptide of this formation particle can comprise the full-length polypeptide of non-sars coronavirus. Perhaps also can use form particle fragment.

In one embodiment, non-SARS particle forms the fragment of polypeptide and the fragment of SARS virus antigen is merged. For example, this chimeric polyeptides can contain born of the same parents' internal area of non-SARS particle formation polypeptide and the extracellular domain of membrane spaning domain and SARS virus antigen. In one embodiment, VLP of the present invention comprises born of the same parents' internal area of containing MHV (MHV) spike protein and the chimeric spike protein of membrane spaning domain, and described chimeric spike protein also contains the extracellular domain of SARS spike protein. This VLP also contains M albumen and the E albumen of coronavirus. Described M and E albumen can be selected from any coronavirus, comprise MHV (MHV) or SARS. The sample sequence that comprises MHV S, M and E albumen among the figure, as above.

The chimeric spike protein from feline infectious peritonitis virus (FIPV) spike protein extracellular domain that is fused to MHV spike protein born of the same parents' internal area and membrane spaning domain is revealed. Referring to WO 98/49195 and WO 02/092827. In these chimeric VLP structures, the M of MHV and E albumen have formed the capsid structure of VLP. Chimeric spike protein makes the extracellular domain of FIPV spike protein be exposed to the surface.

Term " virus-like particle " or " VLP " refer to the virus coat of the non-sky that copies here. VLP is made of one or more virus proteins usually, is called as the protein of capsid protein, coat protein, glutelin, surface protein and/or envelope protein or the granulopotent polypeptide of these protein such as, but not limited to, those. Can spontaneous formation VLP by recombinant expressed these protein in suitable expression system. Perhaps, virus structural protein is separable prepares from intact virus and with phosphatide. The method of making particle VLP is known in the art and below in more detail narration. Can come whether to exist in the detection composition VLP with routine techniques known in the art, these technology such as Electronic Speculum detection, x-radiocrystallography etc. Referring to, for example, Baker etc., Biophys.J. (1991) 60:1445-1456; Hagensee etc., J.Virol. (1994) 68:4503-4505. For example, can be to being carried out electron cryomicroscopy by the aqueous specimen of the VLP goods to be checked of vitreous, and under suitable exposure condition recording picture.

The phrase polypeptide of particle " form " comprises total length or near the virus protein of total length, and fragment, or the virus protein with inner disappearance, and this protein can be conducive to form VLP under the condition that VLP forms. Therefore, described polypeptide can comprise the sequence of full length sequence, fragment, brachymemma and analog and the prerequisite form of partial sequence and mentioned molecule. Therefore, this term comprises to be deleted, adds and replace sequence, as long as this polypeptide still can form VLP. Since the variation of coat protein occurs between the viral isolates usually, so this term comprises the natural variant of specific polypeptide. This term also is included in the mentioned protein and can does not abiogenously lack, add and replace, as long as this protein remains can form VLP.

The preferred replacement is those replacements conservative under natural situation, the replacement that relates to its side chain that namely occurs in class of amino acid. Specifically, amino acid can be divided into 4 classes usually: (1) acidic amino acid: aspartic acid and glutamic acid; (2) basic amino acid: lysine, arginine and histidine; (3) nonpolar amino acid: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar amino acid: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are classified as aromatic amino acid sometimes. For example, can rationally estimate, replace leucine with isoleucine or valine separately, replace aspartic acid with glycine, replace threonine with serine, or replace a seed amino acid with the amino acid conservative of structurally associated, will can not make a big impact to biologically active. Have almost identical amino acid sequence with mentioned molecule, the protein of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor can not affect the immunogenicity of protein substantially but have seldom, so is also contained within the mentioned polypeptide scope.

VLP of the present invention can be formed by any following substances: virus protein, from combination or its fragment of the polypeptide that forms particle or the virus protein of virus protein, they can form particle under proper condition. Requirement for the virus protein that forms particle is, to form in the host cell kytoplasm such as fruit granule, then this protein must be enough stable in the host cell of expressing it, could form viral spline structure like this, and polypeptide will could spontaneously be assembled into viral spline structure in the cell of used recombinant expression system. If protein is secreted in the culture medium, can regulates condition of culture VLP can be formed. In addition, the protein that forms particle should not have cytotoxicity in expressive host, and can not copy in the host that will use VLP.

The preferred polypeptide that forms particle comprises coronavirus M and E albumen, M and the E albumen of preferred SARS.

The method and the appropraite condition that form particle from various virus proteins are known in the art. For example, can make VLP from the protein in following source: influenza virus (such as HA or NA), hepatitis B (such as core protein or capsid protein), HEV, measles virus, sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, norwalk virus, HPV, HIV, the RNA bacteriophage, Q phagus beta (such as coat protein), GA bacteriophage, the fr bacteriophage, AP205 bacteriophage, and Ty (such as retrotransposon Ty albumen p1). VLP further is described in WO 03/024480, and WO 03/024481, and Niikura etc., Virology (2002)293: 273-280; Lenz etc., J.Immunology (2001) 5246-5355; Pinto etc., J.InfectiousDiseases (2003)188: 327-338; With Gerber etc., J.Virology (2001)75(10)：4752-4760。

As mentioned above, VLP can spontaneously form when the protein of interested formation particle is recombinant expressed in suitable host cell. Therefore, being used for VLP of the present invention can be with recombinant technique manufacturing well known in the art. At this moment, can use known technology from the DNA library or directly separate the gene that mentioned coding forms the polypeptide of particle from the cell that contains them with tissue. Also can be based on known array by the synthetic gene of making the polypeptide of coding formation particle. Can design contain suitable codon nucleotide sequence to obtain required specific amino acid sequence. Usually, select to be used for to express the codon (for example the human DNA vaccine being selected human codon) of host's preference of this sequence. Complete sequence is normally assembled from the overlapping oligonucleotides by the standard method preparation, and is assembled into complete coded sequence. Referring to, for example, Edge, Nature (1981) 292:756; Nambair etc., Science (1984) 223:1299; Jay etc., J.Biol.Chem. (1984) 259:6311.

In case it is separated or synthetic that required particle forms the coded sequence of polypeptide, just they can be cloned into any suitable carrier or replicon to express. Be proficient in the known many cloning vectors of technical staff in this field, selecting suitable cloning vector is a selection job. Usually can be referring to the works of Sambrook etc. Then carrier is used to transform suitable host cell. Suitable expression system includes, but not limited to bacterium well known in the art, mammal, baculoviral (bacuolvirus)/insect, cowpox, Semliki forest virus (SFV), yeast and Africa xenopus expression system.

Many clones that are suitable as to make the host cell of VLP of the present invention are known in the art. Suitable mammal cell line comprises, but be not limited to, Chinese hamster ovary (CHO) cell, HeLa cell, baby hamster kidney cell (BHK) cell, MK cells (COS), human liver cell cancer cell (for example, Hep G2), Madin-Darby ox kidney (" MDBK ") cell and other cell. The mammal source of cell comprises, but be not limited to, the mankind or non-human primate are (for example, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), human embryonic kidney cell's (293 cells, usually the adenovirus type 5DNA that is sheared transforms), monkey kidney Vero cell (comprising the COS7 cell), horse, ox (such as the MDBK cell), sheep, dog (dog kidney mdck cell for example, ATCC CCL34MDCK (NBL2) or MDCK 33016, be DSM ACC 2219 such as deposit number as described in the WO 97/37001), cat and mouse (hamster cell for example, such as BHK21-F, HKCC cell or Chinese hamster ovary cell (Chinese hamster ovary celI)), and can available from each stage of development, comprise for example adult, neonate, fetus or embryo.

The bacterial host that is fit to manufacturing VLP of the present invention comprises Escherichia coli (E.coli), bacillus subtilis (Bacillus subtilis) and streptococcus. The yeast host that is fit to manufacturing VLP of the present invention comprises saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), maltose Candida (Candida maltosa), Hansenula polymorpha (Hansenula polymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Pichia guillerimondii, pichia pastoris phaff (Pichia pastoris), fission yeast (Schizosaccharomyces pombe) and Yarrowia lipolytica (Yarrowia lipolytica). The insect cell that is fit to production VLP of the present invention comprises Aedes aegypti (Aedes aegypti), autographa california (Autographa californica), silkworm (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and broccoli looper (Trichoplusia ni).

Viral vectors can be used to make particle in eukaryotic, for example those comprise vaccinia virus and fowlpox virus from the virus of Poxviridae. In addition, based on the bovine vaccine of infection/transfection system, such as Tomei etc., J.Virol (1993) 67:4017-4026 and Selby etc., those described in J.Gen.Virol. (1993) 74:1103-1113 also can be used to produce VLP of the present invention. In this system, at first be encoded vaccinia virus recombinant's in-vitro transfection of T7 phage rna polymerase of cell. This polymerase is only transcribed the template with the T7 promoter. Use the interested DNA transfection of T7 promoters driven after the cell infection. Expression is transcribed into RNA at intracytoplasmic polymerase from vaccinia virus recombinant with the DNA of transfection, and then RNA is translated into protein by host's translating mechanism. The method can be made a large amount of RNA and translation product thereof in high-level ground momently in kytoplasm.

According to selected expression system and host, can be expressed the growth under the polypeptide that forms particle is expressed and can the condition of VLP of host cell that carrier transforms and make VLP by making. Selection to suitable growth conditions is be proficient in this field known to the skilled. If VLP forms in cell, then to destroy cell with chemistry, physics or mechanical means, these methods will make lysis but keep VLP substantially complete. This method is to be proficient in the technical staff in this field and to be described in, for example, " protein purification application practice method " (Protein Purification Applications:A Practical Approach), (E.L.V.Harris and S.Angal compile, 1990).

Then be isolated with the method that keeps particle integrity, as passing through gradient centrifugation, for example, strontium chloride (CsCl) gradient and saccharose gradient, etc. (referring to, for example, Kirnbauer etc., J.Virol. (1993) 67:6929-6936); Ion-exchange chromatography (comprises anion-exchange chromatography, such as DMAE and TMAE), hydroxyapatite (referring to WO 00/09671), hydrophobic interaction chromatography, gel permeation chromatography and other filter method filter and ultrafiltration such as nanometer (nanometric). Preferably in purge process, carry out at least one times anion exchange step, more preferably use at least twice anion exchange step.

Also can use the high concentration reducing agent further to process VLP preparation of the present invention by methods known in the art and make it go to be assembled into the less part that contains protein, and then ressemble CLP by removing reducing agent or adding excessive oxidant. The VLP that gained reassemblies can have improved uniformity, stability and immunogenicity. In addition, can in VLP, add by reassemblying other therapeutic agent or prophylactic. Referring to McCarthy etc., J.Virology (1998) 72 (1): 32-41. Also can be referring to WO 99/13056 and WO 01/42780. The reducing agent that is fit to make VLP go to assemble comprises SH-group reductant (such as glutathione, β mercaptoethanol, dithiothreitol (DTT), the red moss of two sulphur Tang alcohol, cysteine, hydrogen sulfide and their mixture), and they should be contained in ionic medium intensity to the buffer solution of LIS. VLP need to be exposed to the sufficiently long time of reducing agent so that an amount of VLP goes assembling.

Can in VLP of the present invention, add adjuvant with the immunogenicity of enhanced SAR S viral antigen. The antigen that is suitable for VLP comprise above-mentioned those. For example, VLP of the present invention can be adsorbed on the aluminium adjuvant.

VLP of the present invention can be through preparation to strengthen its stability. Other component that can strengthen the VLP preparation stability comprises salt, buffer solution, non-ionic surface active agent and other stabilizing agent, such as the polyanion stabilizing agent of polymerization. Referring to WO 00/45841.

Can keep containing by salt the ionic strength of the solution of VLP particle. The nearly all salt that can control ionic strength all can use. The salt that preferably can be used to regulate ionic strength comprises physiologically acceptable salt, such as NaCl, KCl, Na₂SO ₄、(NH ₄) ₂SO ₄, sodium phosphate and natrium citricum. The concentration of salt component is preferably about 0.10M to 1M. Because restriction uses high salt concentration to carry out parenteral injection in the practice, so very high concentration is not preferred. On the contrary, very moderate salinity, such as physiological concentration preferably, for example about 0.15M is preferred to about 0.5M (wherein containing 0.15M-0.32M NaCl).

Buffer solution also can strengthen the stability of VLP preparation of the present invention. Buffer solution should make the optimal stability of VLP keep simultaneously certain pH scope to make bacterin preparation non-stimulated to the recipient. Buffer solution should maintain the pH of bacterin preparation the scope of pH 5.5-7.0, more preferably 6.0-6.5. The buffer solution that is suitable for bacterin preparation is known in the art, comprising, for example, histidine and imidazoles. The concentration range of buffer solution is preferably about 2mM to about 100mM, more preferably 5mM-20mM. When VLP was adsorbed by aluminium compound or prepares with aluminium compound with other method, containing phosphatic buffer solution was not preferred usually.

Nonionic surfactant can be used to strengthen the stability of VLP preparation of the present invention. The surfactant that is suitable for bacterin preparation is known in the art, comprising, for example, polyoxyethylene sorbitan fatty acid ester (polysorbate class), such as polysorbate80 (for example, TWEEN 80), polysorbate20 (for example, TWEEN 20), polyoxyethylene alkyl ether (for example, Brij 35, and Brij 58), the non-ionic surface active agent (for example, Pluronic 121) that comprises in addition Triton X-100, Triton X-114, NP-40, Span 85 and Pluronic series. The concentration of surfactant preferably is about 0.0005% to about 0.5% (weight per volume).

The polyanion stabilizing agent of polymerization can be used to strengthen the stability of VLP preparation of the present invention. The polyanion stabilizing agent that is applicable to polymerization of the present invention contains single long-chain or a plurality of crosslinked chain; When in solution, on the chain of any type all with a plurality of negative electrical charges. Suitable polyanionic polymer comprises protein, polyanion, peptide class and polynucleic acid. Concrete example comprises glutathione, polynucleotides, RNA, DNA and the seralbumin of carboxymethyl cellulose, heparin, polyaminoacid (such as poly-(Glu), poly-(Asp) and poly-(Glu, Phe)), oxidation. The concentration of the polyanion stabilizing agent of polymerization is preferably about 0.01% to about 0.5%, especially about 0.05-0.1% (percentage by weight).

G. the antibody passive immunity by SARS antigen of the present invention

The present invention includes the specific antibody of SARS antigen of the present invention and the method for the treatment of or preventing the SARS virus relevant disease by the SARS antibody that gives the mammalian object effective dose. The specific antibody of SARS virus can be made by the technical staff who is proficient in this field. Preferred described antibody is the specific antibody of SARS virus furcella (S) albumen. Reported with human monoclonal and resisted-furcella antibody efficient neutralization sars coronavirus (Sui etc., (2004) PNAS USA 101:2536-2541). The IgG1 form of monoclonal antibody demonstrates the affinity (1.59nM) that is higher than scFv form (32.3nM).

Antibody of the present invention is specificity and optionally for SARS antigen.

In one embodiment, antibody of the present invention produces by use SARS antigen to animal. The method also comprises from the animal separation antibody.

Antibody of the present invention can be polyclone or monoclonal antibody goods, monospecific antiserum, and human antibodies can be hybrid antibody or chimeric antibody perhaps, such as humanized antibody, variation antibody (Fab ')₂Fragment, F (ab) fragment, Fv fragment, single domain antibody, dimerization or trimerization antibody fragment or construction, microbody, or its functional fragment of being combined with mentioned antigen.

Antibody is to make with the technology known by the technical staff of being proficient in this field, and these technical descriptions exist, for example, and United States Patent(USP) Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; In 3,770,380 and 4,372,745. For example, polyclonal antibody is by producing with the suitable animal of interested antigen immune (such as mouse, rat, rabbit, sheep or goat). For strengthening immunogenicity, can antigen be attached on the carrier then in immunity first. This carrier is that one of ordinary skill in the art is known. Immunity is carried out usually like this: antigen is mixed or emulsification with salt solution (better with adjuvant such as Freund's complete adjuvant), and then parenteral (normally subcutaneous or intramuscular) is injected this mixture or emulsion. The antigen injection animal one or many booster immunization of using afterwards salt solution (preferably using incomplete Freunds adjuvant) to join in 2-6 week. Also can carry out external immunity with methods known in the art and produce antibody. Then the animal from immunity obtains polyclonal antiserum.

Method [(1975) Nature with Kohler and Milstein256: 495-497] or its improve one's methods and make monoclonal antibody. Usually, as mentioned above to mouse or rat immunity. Also can use rabbit. Yet, be not to be animal to be got blood extract serum, but take out spleen (and randomly taking out several large lymph nodes), it is dispersed into unicellular. If necessary, cell suspension (after removing the cell of non-specific adhesion) adding can be coated with in the plate or hole of proteantigen, splenocyte has been screened. Express the B Cell binding of membrane bound immunoglobulin of antigentic specificity to plate, do not resemble other material of suspension by flush away. Then gained B cell or separative splenocyte are induced, make itself and myeloma cell merge the formation hybridoma, cultivate in selective medium (such as hypoxanthine, aminopterin, thymidine culture medium, " HAT "). By limiting dilution inoculation gained hybridoma, and the generation of the antibody of mensuration specific binding immunizing antigen (and not in conjunction with irrelevant antigen). Then, the hybridoma of the selected secrete monoclonal antibody of (in mouse ascites) cultivation in external (for example in tissue culture flasks or hollow fiber reactor) or the body.

Humanized antibody and chimeric antibody also can be used for the present invention. Hybridization (chimeric) antibody molecule is described in Winter etc., (1991) Nature usually349: 293-299 and U.S. Patent No. 4,816,567. Humanized antibody molecules is described in Riechmann etc. usually, (1988) Nature332: 323-327; Verhoeyan etc., (1988) Science239: 1534-1536; With UK Patent Application No.GB 2,276,169 (on September 21st, 1994 is open). The method that a kind of engineering makes up humanized antibody comprises that the clone contains the recombinant DNA of promoter, leader and mouse antibodies gene variable region sequences and human immunoglobulin gene constant region extron, to produce mouse-people's chimera, i.e. humanized antibody. Usually can be referring to Kuby, " immunology " (Immunology), the third edition, W.H.Freeman and Company, New York (1998), the 136th page.

The antibody fragment that can identify SARS antigen is also included within the scope of the invention. Many antibody fragments that contain the antigen binding site that can show complete antibody molecular immune binding characteristic are known in the art. For example, can be by coming the manufacturing function antibody fragment as producing F (ab ') with Pepsin digestion for example except the constant region of not responsible antigen combination in the antibody molecule₂Fragment. These fragments will contain two antigen binding sites, but lack the part constant region of every heavy chain. Similarly, if necessary, can make the Fab fragment that contains single antigen binding site, for example, by using papain digestion polyclone or monoclonal antibody. Also but the Application standard technology is made the functional fragment of the variable region that only comprises heavy chain and light chain, these technology such as recombination method or preferential proteolysis cutting immunoglobulin molecules. These fragments are called as F_v Referring to, for example, Inbar etc., (1972) Proc.Nat.Acad.Sci USA69: 2659-2662; Hochman etc., (1976) Biochem15: 2706-2710; And Ehrlich etc., (1980) Biochem19：4091-4096。

ScFv (" sFv " or scFv ") polypeptide is a kind of covalently bound V_H-V _LHeterodimer, it is the V that connects by comprising peptide coding joint_H-and V_LThe fusion of-encoding gene is expressed. Huston etc., (1988) Proc. Nat.Acad.Sci.USA85: 5879-5883. Describe many evaluations and set up the method for chemical constitution (joint), these structures are used for natural cohesion but light chain and the heavy chain polypeptide in the antibody V zone that can separate by chemical method change the sFy molecule into, and the sFv molecule will be folded into and the basic similarly three-dimensional structure of structure of antigen binding site. Referring to, for example, United States Patent(USP) Nos. 5,091,513; 5,132,405; With 4,946,778. Can be used on the method for having described this area and make the sFv molecule. Referring to, for example, Huston etc., (1988) Proc.Nat. Acad.Sci USA85: 5879-5338; United States Patent(USP) Nos. 5,091,513; 5,132,405 and 4,946,778. Design standard comprises the substantial distance of distance between the N-end of terminal and another chain of the C-that determines a chain, and wherein said joint is formed by the little hydrophilic amino acid residue that can not be curling or forms secondary structure usually. This method was described in this area. Referring to, for example, United States Patent(USP) Nos. 5,091,513; 5,132,405 and 4,946,778. Suitable joint generally includes the polypeptide chain that glycine and serine can be replaced mutually, and can comprise and insert glutamic acid and lysine residue to strengthen dissolubility. Anti--furcella scFv antibody be in the news (Sui etc., (2004) PNAS USA 101:2536-2541).

" small antibody (Mini-antibody) " or " microbody (Minibody) " also can be used for the present invention. Microbody is the sFv polypeptide chain, comprises the oligomerization zone of separating by hinge region and sFv at their C-end. Pack etc., (1992) Biochem31: 1579-1584. The oligomerization zone comprises that for example leucine zipper can make it more stable by other disulfide bond from uniting alpha-helix. The oligomerization zone is designed to folding consistent with the cross-film directionality, and this process is considered to be conducive to be folded in the polypeptide body functional in conjunction with albumen. Usually, microbody is made with recombination method well known in the art. Referring to, for example, Pack etc., (1992) Biochem31: 1579-1584; Cumber etc., (1992) J.Immunology149B：120-126。

Also can produce and identify antibody of the present invention with nconventional method. For example, but the screening phage display library can be in conjunction with the antibody of SARS antigen of the present invention. Usually can be referring to Siegel, " recombinant monoclonal antibodies technology " (Recombinant Monoclonal Antibody Technology), Transfus.Clin.Biol. (2002)9(1): 15-22; Sidhu, " phage display is used for Pharmaceutical Biotechnology " (Phage Display in Pharmaceutical Biotechnology), Curr.Opin.Biotechnol. (2000)11(6): 610-616; Sharon etc., " recombinant polyclonal antibody library " (Recombinant Polyclonal Antibody Libraries), Comb.Chem.High Throughput Screen (2000)3(3): 185-196; With Schmitz etc., " phage display: produce the molecular tool of antibody-review " (Phage Display:A Molecular Tool for the Generation of Antibodies-Review), Placenta, (2000)21 SupplA：S106-12。

Can make antibody of the present invention by the polynucleotide sequence that gives animal encoding SARS antigen. Then SARS antigen express in vivo, and generate in vivo the SARS antigen-specific antibodies. Carry the method for SARS antigen of the present invention with below the 4th part discussion with polynucleotides.

Antibody of the present invention should be specific to SARS virus.

H. the combination of one or more above-mentioned vaccines

Composition of the present invention also comprises the combination of one or more above-mentioned compositions. For example, the present invention includes the composition that contains attenuation SARS virus and subunit's SARS virus antigen.

The combination of L.SARS antigen and other Respirovirus antigen

The invention still further relates to the bacterin preparation that contains one or more SARS virus antigens and one or more other Respirovirus antigens. Be applicable to other Respirovirus antigen of the present invention and comprise antigen from lower influenza virus: influenza virus, ERC group virus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus(RSV) (RSV), adenovirus, super Pneumovirinae and rhinovirus. Other Respirovirus antigen can be from the coronavirus outside the sars coronavirus, such as NL63 human corona virus (van der Hoek etc., (2004) Nature Medicine 10:368-373). Preferred other Respirovirus antigen is influenza antigen.

The present invention also comprises one or more bacteriums or the viral antigen with the combination of SARS virus antigen. Described antigen can use separately or be used in combination by any way. (referring to, for example, WO 02/00249, wherein described the application of bacterial antigens combinations). This composition can comprise the plurality of antigens from same pathogen, from a plurality of antigens of different pathogens or from identical and a plurality of antigens different pathogens. Therefore, bacterium, virus and/or other antigen can be contained in the same composition, perhaps can give respectively same target. The antigen that usually should will be used for causing immune response is used in combination.

The non-limitative example that can be used for bacterial pathogens of the present invention comprises: diphtheria (referring to, for example, " vaccine " the 3rd chapter, 1998, Plotkin and Mortimer compile (ISBN 0-7216-1946-0), staphylococcus (for example, [(2001) Lancet 357:1225-1240] the described staphylococcus aureuses such as Kuroda (Staphylococcus aureus)), cholera, tuberculosis, clostridium tetani (C.tetani), be also referred to as lockjaw (referring to, for example, " vaccine " the 4th chapter, 1998, Plotkin and Mortimer compile (ISBN 0-7216-1946-0), A group and B group of streptococcus (comprise streptococcus pneumonia (Streptococcus pneumoniae), Streptococcusagalactiae (Streptococcus agalactiae) and streptococcus pyogenes (Streptococcuspyogenes), are described in such as Watson etc., (2000) Pediatr.Infect.Dis.J.19:331-332; Rubin etc., (2000) Pediatr Clin.North Am. 47:269-284; Jedrzejas etc., (2001) Microbiol Mol Biol Rev 65:187-207; Schuchat (1999) Lancet 353:51-56; BP patent application 0026333.5; 0028727.6; 015640.7; Dale etc., (1999) Infect Dis Clin North Am 13:227-1243; Ferretti etc., (2001) PNASUSA 98:4658-4663), pertussis (referring to, for example, Gusttafsson etc., (1996) N.Engl.J.Med.334:349-355; Rappuoli etc., (1991) TIBTECH9:232-238), meningitis, Moraxella catarrhalis (Moraxella catarrhalis) (referring to, for example, McMichael (2000) Vaccine 19 Suppl.1:S101-107) and the Other diseases state, comprising, but be not limited to, Neisseria meningitidis (Neisseria meningitides) (A, B, C, Y), NEISSERIA GONORRHOEAE (Neisseria gonorrhoeae) is (referring to for example WO 99/24578; WO 99/36544; With WO 99/57280), (for example, CagA, VacA, NAP, HopX, HopY and/or urase are such as for example WO 93/18150 for helicobacter pylori (Helicobacter pylori); WO 99/53310; WO 98/04702 is described) and haemophilus influenzae (Haemophilus influenza). B type haemophilus influenzae (HIB) is (referring to such as Costantino etc., (1999) Vaccine 17:1251-1263), porphyromonas gingivalis (Porphyromonas gingivalis) (Ross etc., (2001) Vaccine 19:4135-4132) and combination thereof.

The non-limitative example that can be used for viral pathogen of the present invention comprises: meningitis, rhinovirus, influenza (Kawaoka etc., Virology (1990) 179:759-767; Webster etc., " antigenic variation of influenza A virus " (Antigenic variation among type A influenza viruses), the 127-168 page or leaf, close at P.Palese and D.W.Kingsbury (volume), " influenza virus science of heredity " (Genetics of influenza viruses. Springer-Verlag, New York), Respiratory Syncytial Virus(RSV) (RSV), parainfluenza virus (PIV), rotavirus (for example, VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 or NSP5, and other wheel virus antigen, as described in WO 00/26380), etc. Also can be used for the present invention derived from other viral antigen, nonrestrictive example comprises the protein from following Viraceae member: Picornaviridae (such as poliovirus etc., is described in such as (2000) Pediatr Clin North Am 47:287-308 such as Sutter; Zimmerman and Spann (1999) Am Fam Physician 59:113-118; 125-126); Caliciviridae; Togaviridae (such as rubella virus etc.); Flaviviridae comprises common flavivirus (for example, yellow fever virus, japanese encephalitis virus, the serotype of dengue virus, tick-brone encephalitis virus, west nile virus, St. Louis encephalitis virus); Pestivirus (for example, classic pig heating virus, bovine viral diarrhea virus, border disease virus); And HCV (for example United States Patent(USP) Nos. 4,702, and 909; 5,011,915; 5,698,390; 6,027,729 and 6,297, the first type described in 048, B-mode and hepatitis C); Tiny parvovirus (for example assays for parvovirus B 19); Coronaviridae; Reoviridae; Bimaviridae; Rhabdoviridae (such as rabies viruses etc., is described in (1997) the Vaccine 15Suppl:s2-6 such as Dressen; MMWR Morb Mortal Wkly Rep.1998Jan 16:47 (1): 12,19); Filamentous form virus section; Paramyxoviridae (for example, mumps virus, measles virus, Respiratory Syncytial Virus(RSV) etc., of " vaccine " [1998, Plotkin and Mortimer compile (ISBN 0-7216-1946-0)] 9-11 chapter; Orthomyxoviridae family (for example, A, B and C type influenza virus etc., of " vaccine " [1998, Plotkin and Mortimer compile (ISBN 0-7216-1946-0)] the 19th chapter); Bunyaviridae; Arenaviridae; Retroviridae (for example, HTLV-1; HTLV-11; HIV-1 (being also referred to as HTLV-III, LAV, ARV, HTI, R etc.)), include but not limited to the antigen of HIVIllb, HIVSF2, HIVLAV, HIVI-AL, I-IIVMN, SF162 separator); HIV-I CM235, HIV-I US4; HIV-2; SIV (SIV). In addition, but antigen also derived from human papillomavirus (HPV) and tick-brone encephalitis virus. For understanding relevant these virus and other viral narrations, can be referring to for example " virology " (Virology), the third edition (W.K.Joklik compiles, 1988); " basic virology " (Fundamental Virology), second edition (B.N.Fields and D.M.Knipe compile, 1991).

Protein can derived from herpetoviridae, comprise the protein derived from 1 type and herpes simplex types 2 virus (HSV), such as HSV-1 and HSV-2 glycoprotein gB, gD and gH; Derived from the antigen of herpes zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), comprise CMV gB and gH (see U.S. Patent No. 4,689,225 and the open W089/07143 of PCT); And derived from other nerpes vinrus hominis's antigen, such as HHV6 and HHV7. (see such as [" cytomegalovirus " be (J.K.McDougall compile, and Springer-Verlag 1990) (Cytomegaloviruses), the 125-169 page or leaf] such as Chee to understand the protein coding content of cytomegalovirus; [the J.Gen. Virol. (1988) such as McGeoch69: 1531-1574] to understand the albumen of various HSV-1 codings; U.S. Patent No. 5,171,568 is to understand HSV-1 and HSV-2gB and gD albumen and their gene of encoding; [the Nature (1984) such as Baer310: 207-211] to identify the genomic protein coding sequence of EBV; And Davison and Scott [J.Gen.Virol. (1986)67: 1759-1816] to understand VZV). Herpes simplex virus (HSV) rgD2 is a kind of recombinant protein that produces through genetic engineering modified Chinese hamster ovary cell. The normal anchorage zone of this protein is caused glycosylated protein to be secreted in the tissue culture medium (TCM) by brachymemma. GD2 in the CHO culture medium can be purified to purity greater than 90%. Human immunodeficiency virus (HIV) env-2-3 is the recombinant forms through the HIV envelope protein of genetic engineering modified saccharomyces cerevisiae (Saccharomyces cerevisae) generation. This protein has the complete protein zone of HIV gp120, but nonglycosylated sex change when purifying from yeast. HIV gpl20 is the complete glycosylated secreted form of gp120, produces in Chinese hamster ovary celI in the similar mode of above-mentioned gD2. Other HSV antigen that is suitable for immunogenic composition is described among the open WO 85/04587 of PCT and the WO 88/02634, and they are included in this paper as a reference in full. The mixture of gB and gD antigen (surface antigen that lacks the brachymemma of anchorage zone) is particularly preferred.

Antigen from following hepatitis viruse section also can be advantageously used in technology described here, and these viruses comprise that hepatitis A virus (HAV) is (referring to such as Bell etc., (2000) Pediatr Infect Dis.J.19:1187-1188; Iwarson (1995) APMIS 103:321-326), hepatitis type B virus (HBV) is (referring to such as Gerlich etc., (1990) Vaccine 8 Suppl:S63-68 ﹠ 79-80), HCV (HCV) (referring to for example PCT/US88/04125, No. the 318216th, disclosed European application), Hepatitis D virus (HDV), HEV (HEV) and HGV RNA (HGV). For example, the virus genome sequence of known HCV is known the method that obtains sequence simultaneously. Referring to, for example, international publication number is the application of WO 89/04669, WO 90/11089 and WO 90/14436. The present invention also comprises the molecular variants of this peptide species, for example PCT/US99/31245; PCT/US99/31273 and PCT/US99/31272 are described. The HCV genome of several virus proteins of encoding comprises that E1 (being also referred to as E) and E2 (being also referred to as E2/NSI) and the terminal nucleocapsid protein of N-(being called " core " (core)) (see [Hepatology (1991) such as Houghton14: 381-388] to understand the narration about HCV albumen, comprise E1 and E2). Similarly, the sequence of HDV δ-antigen also is known (seeing for example U.S. Patent No. 5,378,814), and this antigen can be advantageously used in the compositions and methods of the invention. In addition, can also use the antigen from HBV, also has the combination of above-mentioned antigen such as cAg, surface antigen, SAg and front surface sequence (presurface sequences) pre-S1 and pre-S2 (being called in the past pre-S), such as SAg/pre-S1, SAg/pre-S2, SAg/pre-S1/pre-S2 and pre-S1/pre-S2. For example see, " HBV vaccine one is from the laboratory to the license: analysis of cases " (HBV Vaccines-from the laboratory to license:a case study), be selected from Mackett, M. and Williamson, J.D., " human vaccine and immunity inoculation " (Human Vaccines and Vaccination), the 159-176 page or leaf is to understand the structure of HBV; And United States Patent(USP) Nos. 4,722,840,5,098,704,5,324,513, they are included in this paper as a reference in full; Beames etc., J.Virol. (1995)69: 6833-6838, Birnbaum etc., J.Virol. (1990)64: 3319-3330; With Zhou etc., J.Virol. (1991)65: 5457-5464. These protein and their antigenicity fragment all can be used for the compositions and methods of the invention.

Influenza virus is especially effectively another example of virus of the present invention. Specifically, for producing immune response, envelope glycoprotein HA and the NA of people's special concern Flu-A. Many HA hypotypes of Flu-A are identified (Kawaoka etc., Virology (1990) also179: 759-767; Webster etc., " antigenic variation of influenza A virus " (Antigenic variation among type A influenza viruses), the 127-168 page or leaf, be selected from P.Palese and D.W.Kingsbury (volume), " influenza virus science of heredity " (Genetics of influenza viruses. Springer-Verlag, New York). Therefore, can be used for composition described here and method derived from protein any in these separators.

The non-limitative example of parasite antigen comprises the antigen that those cause the organism of malaria and Lyme disease.

Method of the present invention comprises that giving a kind of SARS virus antigen that contains of animal (comprises one or more deactivations SARS virus, attenuation SARS virus, the recombinant of SARS lytic virus goods or one or more SARS virus antigens or subunit's preparation of purifying) immunogenic composition. Be used for the SARS virus antigen that immunogenic composition of the present invention can comprise immune effective dose. " immune effective dose " refers to be enough to make animal to produce the amount of the immune response of anti-SARS antigen.

Described immune response preferably includes and produces the SARS antigen-specific antibodies. The amount of the antibody that produces will look used animal, whether have the factor such as adjuvant and difference.

Immunogenic composition of the present invention also contains one or more adjuvants.

But immunogenic composition mucous membrane of the present invention medication. That suitable mucous membrane route of administration comprises is oral, in the nose, in the stomach, in the lung, intestines, rectum, eye and vaginal approach. Immunogenic composition can be made into to be fit to the form of mucous membrane medication. For example, when oral use composition, it can be made into the forms such as tablet or capsule (the optional casing that is surrounded by), liquid, genetically modified plants. When said composition was used to intranasal administration, it can be the form of nose spraying, nose dropping liquid, gel or powder.

But immunogenic composition parenteral of the present invention medication. Suitable parenteral route of administration comprises in (IM) in the muscle, subcutaneous, intravenous, the peritonaeum, in the corium, through skin and transdermal (referring to for example WO 98/20734) approach, and is transported to tissue space. Immunogenic composition can be made into to be fit to the form of parenteral medication, for example aseptic or pyrogen-free injectable forms.

Vaccine of the present invention can be co-administered with other immunomodulator. Specifically, composition will comprise adjuvant usually. Other preferred adjuvant includes, but not limited to one or more following substances:

A. the composition that contains mineral matter

The composition that contains mineral matter that is suitable as adjuvant of the present invention comprises mineral salts, such as aluminium salt and calcium salt. The present invention includes following mineral salts, such as (visible " vaccine design: subunit and adjuvant research " (Vaccine design:the subunit and adjuvant approach) the 8th and 9 chapters for example such as hydroxide (such as oxyhydroxide), phosphate (such as hydroxyl phosphate, orthophosphates), sulfate, (1995) Powell and Newman.ISBN 0-306-44867-X.), the perhaps mixture of different minerals matter and described compound, can take any suitable form (such as glue, crystal, amorphous etc.), preferably be adsorbed. The composition that contains mineral matter can be made into the slaine particle. Referring to WO 00/23105.

B. oiliness emulsion

The oiliness emulsion composition that is suitable as adjuvant of the present invention comprises MF59-aqueous emulsion, such as MF59 (5% MF59,0.5% Tween 80 and 0.5%Span 85 make the submicron particle with micro-Fluidizer). Referring to WO90/14837. Also can be referring to Frey etc., " MF59 adjuvant influenza vaccines and without security, tolerance and immunogenicity " (the Comparison of the safety of the influenza vaccines of adjuvant relatively in non-aged adult, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non-elderly adults ", Vaccine (2003)21：4234-4237。

The adjuvant that is particularly preferred for composition is the submicron oil in water emulsion. Be the MF59/aqueous emulsion that randomly contains the MTP-PE of different amounts with the preferred submicron oil in water emulsion here, such as the submicron oil in water emulsion, wherein contain 4-5%w/v MF59,0.25-1.0%w/v Tween 80^TM(polyoxyethylene sorbose monooleate) and/or 0.25-1.0%Span 85^TM(sorbitan trioleate), and the optional N-acetyl muramyl-different glutaminase acyl group-ALANINE of L-alanyl-D--2-(1 '-2 '-two palmityls-sn-glycerine-3-hydroxyl phosphinylidyne oxygen)-ethamine (MTP-PE), (WO 90/14837 for example to be called the submicron oil in water emulsion of " MF59 "; United States Patent (USP) 6,299,884 and 6,451,325, they are included in this paper as a reference in full; With Ott etc., " MF59-design and evaluate safety and effective human vaccine adjuvant " (MF59-Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines), be selected from " vaccine design: subunit and adjuvant research " (Powell, M.F. and Newman, M.J. compile) Plenum Press, New York, 1995,277-296). MF59 contains 4-5% w/v MF59 (for example 4.3%), 0.25-0.5%w/v Tween 80^TMWith 0.5%w/v Span 85^TM, and the optional MTP-PE that contains different amounts, be mixed with the submicron particle with micro-Fluidizer such as 110Y type trace Fluidizer (Microfluidics, Newton, MA). For example, the content of MTP-PE is about 0-500 μ g/ agent, and more preferably 0-250 μ g/ agent most preferably is 0-100 μ g/ agent. Term " MF59-0 " represents not contain the above-mentioned submicron oil in water emulsion of MTP-PE here, and term MF59-MTP represents to contain the preparation of MTP-PE. For example, " MF59-100 " contains 100 μ g MTP-PE/ agent, so analogizes. Another kind of submicron oil in water emulsion MF69 used herein contains 4.3%w/v MF59,0.25%w/v Tween 80^TMWith 0.75%w/v Span 85^TM, and the optional MTP-PE that contains. Another kind of submicron oil in water emulsion is MF75, is also referred to as SAF, contains 10% MF59,0.4 %Tween 80^TM, 5% Pluronic (pluronic) block polymer L121 and thr-MDP, also changed into submicron emulsion by microfluid. MF75-MTP represents to contain the preparation of MTP, for example 100-400 μ g MTP-PE/ agent.

The submicron oil in water emulsion is made this emulsion methods, and is described in detail in WO 90114837 and United States Patent(USP) Nos. 6 for the immunostimulant (such as muramyl peptide) of the present composition, 299,884 and 6,451,325, they are included in this paper as a reference in full.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) also can be used as adjuvant of the present invention.

C. saponin(e preparation

The saponin(e preparation also can be used as adjuvant of the present invention. Saponin(e is a kind of at many floristic barks, leaf, stem, root even spend the allos group of steroline and the triterpene glycosides of middle discovery. Saponin(e from Quillaia saponaria Molina tree bark is widely studied as adjuvant. Saponin(e also can be commercial available from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil) and Saponaria officianalis (Radix saponariae) by passing through. The saponin adjuvant preparation comprises the preparation of purifying, such as QS21, and liquid preparation, such as ISCOM.

Usable highly effective thin-layer chromatography (HP-LC) and RPLC (RP-HPLC) purifying astragalin composition. The concrete purified components of using these technology to obtain is identified, comprises QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferred described saponin(e is QS21. The method of a kind of QS21 of manufacturing is disclosed in U.S. Patent No. 5,057,540. Also can contain sterol in the saponin(e preparation, such as cholesterol (referring to WO 96/33739).

The combination of saponin(e and cholesterol can be used to form the unique particle that is called as immunostimulating complex (ISCOM). ISCOM also contains phosphatide usually, such as phosphatidyl-ethanolamine or phosphatid ylcholine. Any known saponin(e all can be used for ISCOM. Preferred ISCOM comprises one or more among Quil A, QHA and the QHC. ISCOM also is described in EP 0 109942, WO 96/11711 and WO 96/33739. Randomly, ISCOM can not contain other detergent. Referring to WO00/07621.

For the manufacturing of understanding saponin(e base adjuvant can be referring to Barr etc., and " ISCOM and other saponin(e base adjuvant " (ISCOMs and other saponin based adjuvants ", Advanced Drug Delivery Reviews (1998)32: 247-271. Also can be referring to Sjolander etc., " picked-up of oral saponin(e and ISCOM vaccine and adjuvanticity " (Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines ", Advanced Drug Delivery Reviews (1998)32：321-338。

D. bacterium or microorganism derivative

Be applicable to adjuvant of the present invention and comprise bacterium or microorganism derivative, as:

(1) non-toxic derivant of enteric bacteria lipopolysaccharides (LPS)

This derivative comprises monophosphoryl lipid A (MPL) and 3-O-deacylated tRNA MPL (3dMPL). 3dMPL is the mixing of 3-O-deacylated tRNA monophosphoryl lipid A and 4,5 or 6 acidylate chains. " granule " form of preferred 3-O-deacylated tRNA monophosphoryl lipid A is disclosed in EP 0 689 454. This " granule " of 3dMPL is enough little so that can pass through 0.22 micron aseptic filter membrane (referring to EP 0 689 454). Other nontoxic LPS derivative comprises the monophosphoryl lipid A analogies, such as aminoalkyl glucosaminide phosphoric acid derivatives, for example RC-529. See Johnson etc., (1999) BioorgMed Chem Lett 9:2273-2278.

(2) lipid A derivative

The lipid A derivative comprises from colibacillary lipid A derivative, such as OM-174. OM-174 is described in; for example; Meraldi etc.; " a kind of new might be used for human adjuvant OM-174 after the C-terminal fragment 242-310 of synthetic plasmodium berghei circumsporozoite protein uses, induce protective response " (OM-174; a New Adjuvant with a Potential for Human Use; Induces a Protective Response with Administered with the Synthetic C-Terminal Fragment 242-310 from the circumsporozoite protein of Plasmodium berghei), Vaccine (2003)21: 2485-2491; With Pajak etc., " the OM-174 adjuvant is induced migration and the maturation of mouse dendritic cells in vivo " (The Adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo ", Vaccine (2003)21：836-842。

(3) immunostimulatory oligonucleotide

The immunostimulatory oligonucleotide that is suitable as adjuvant of the present invention comprises the nucleotide sequence that contains CpG motif (one section contains after the unmethylated cytimidine is guanine and the sequence that connects by phosphate bond). Proved that the bacterium double-stranded RNA or the oligonucleotides that contain palindromic sequence or poly-(dG) sequence have immunostimulatory activity.

CpG can comprise nucleosides modification/analog, modifies such as phosphorothioate ester, and can be two strands or strand. Randomly, guanosine can be substituted such as 2 '-deoxidation-7-denitrogenation guanosine by its analog. Such as can be referring to [" divergent synthetic nucleotides motif recognition mode: design and set up the few DNA preparation of the potent immune regulative with different cytokines inducing properties " (Divergent synthetic nucleotide motif recognition pattern:design and development of potent immunomodulatory oligodeoxy ribonucleotide agents with distinct cytokine induction profiles), the Nucleic Acids Research (2003) such as Kandimalla31(9): 2393-2400]; WO 02/26757 and WO 99/62923 have narrated possible analog example. The adjuvant effect of CpG ODN also is described in Krieg, " CpG motif: the active component in the bacterial extract? " (CpG motifs:the active ingredient in bacterial extracts?), Nature Medicine (2003) 9 (7): 831-835; McCluskie etc., " in mouse, use the strategy of hepatitis B surface antigen and CpG DNA booster immunization by parenteral and mucous membrane " (Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA), FEMS Immunology and Medical Microbiology (2002)32: 179-185; WO 98/40100; U.S. Patent No. 6,207,646; U.S. Patent No. 6,239,116 and U.S. Patent No. 6,429,199.

The orientable TLR9 of CpG sequence is such as GTCGTT or TTCGTT motif. See Kandimalla etc., " Toll sample receptor 9: regulate evaluation and cytokine induction by new synthetic CpG DNA " (Toll-like receptor 9:modulation of recognition and cytokine induction by novel synthetic CpG DNAs), Biochemical Society Transactions (2003)31(the 3rd part): 654-658. The CpG sequence can be specific to induces the Th1 immune response, and such as CpG-A ODN, perhaps it may more be specific to and induce the B cell response, such as CpG-B ODN. CpG-A and CpG-B ODN are described in Blackwell etc., " but the IFN-α adjusting that monocyte IFN-γ-induced protein that CpG-A induces is derived by plasmacytoid dendritic cells " (CpG-A-Induced Monocyte IFN-gamma-Inducible Protein-10 Production is Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha), J.Immunol. (2003)170(8)： 4061-4068；Krieg，“CpG A-Z”(From A to Z on CpG)，TRENDS in Immunology(2002) 23(2): 64-65 and WO 01/95935. Preferred described CpG is CpG-A ODN.

Preferred CpG ODN be make up so that 5 ' end can be by Receptor recognition. Randomly, two CpG ODN sequences can be in their 3 ' terminal linking to each other with formation " immunity (immunomer) ". Referring to such as Kandimalla etc., " affect the secondary structure of the CpG ODN of immunostimulatory activity " (Secondary structures in CpG oligonucleotides affect immunostimulatory activity), BBRC (2003)306: 948-953; Kandimalla etc., " Toll sample receptor 9: regulate evaluation and cytokine induction by new synthetic CpG DNA ", Biochemical Society Transactions (2003)31(the 3rd part): 664-658; Bhagat etc., " CpG five-and six DNAs are effective immunomodulators " (CpG penta-and hexadeoxyribonucleotides as potent immunomodulatory agents) BBRC (2003)300: 853-861 and WO 03/035836.

(4) ADP ribosylation toxin and detoxification derivative thereof

Bacterium ADP ribosylation toxin and detoxification derivative thereof can be used as adjuvant of the present invention. Preferred described protein derived is from Escherichia coli (be E.coli LT " LT), cholera (" CT ") or pertussis (" PT "). WO 95/17211 has described use detoxification ADP ribosylation toxin as mucosal adjuvants, and WO 98/42375 has described it as the parenteral adjuvant. Preferred adjuvant is the 192-GLY LT of detoxification, such as LT-K63, LT-R72 and LTRl92G. Can in below with reference to data, find ADP ribosylation toxin and detoxification derivative thereof, especially LT-K63 and LT-R72, as adjuvant, fit into this paper as a reference in these lists of references: Beignon etc., " the LTR72 mutant of E.coli LT is applied to the ability that strengthens peptide antigen stimulation CD4+T cell and secrete IFN-γ behind the Bare skin altogether " (The LTR72 Mutant of Heat-Labile Enterotoxin of Escherichia coli Enahnces the Ability of Peptide Antigen to Elicit CD4+T and Secrete Gamma Interferon after Coapplication onto Bare Skin), Infection and Immunity (2002) 70 (6): 3012-3019; Pizza etc., " mucosal vaccine: as the LT of mucosal adjuvants and the non-toxic derivant of CT " (Mucosal vaccines:non-toxic derivatives of LT and CT as mucosal adjuvants), Vaccine (2001)19: 2534-2541; Pizza etc., " LTK63 and LTR72, two kinds will be carried out the mucosal adjuvants of clinical testing " (LTK63and LTR72, two mucosal adjuvants ready for clinical triais) Int.J.Med.Microbiol (2000)290(4-5): 455-461; Scharton-Kersten etc., " with bacterium ADP ribosylation exotoxin, subunit and irrelevant adjuvant transcutaneous immune " (Transcutaneous Immunization with Bacterial ADP-Ribosylat ing Exotoxins, Subunits and Unrelated Adjuvants), Infection and Immunity (2000)68(9): 5306-5313; Ryan etc., " mutant of HLT is carried the potent mucosal adjuvant of acellular pertussis vaccine as intranasal: the not same-action of nontoxic AB complex and the enzymatic activity on Th1 and Th2 cell " (Mutants of Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine:Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Th1 and Th2 Cells) Infection and Immunity (1999)67(12): 6270-6280; Partidos etc., " E.coli LT and directed mutants LTK63 thereof strengthen the altogether breeder reaction of immune synthetic peptide and cytotoxic T cell reaction in the nose " (Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides), Immunol. Lett. (1999)67(3): 209-216; Peppoloni etc., " the E.coli LT mutant is to carry the effective adjuvant of vaccine safety in the nose " (Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines), Vaccines (2003) 2 (2): 285-293; With Pine etc., (2002) " with immunity in detoxification mutant (LTK63) nose of influenza vaccines and E.coli LT " (Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxin from Escherichia coli (LTK63)) J.Control Release (2002) 85 (1-3): 263-270. Amino acid replacement is preferably based on the sequence contrast of ADP ribosylation toxin A and B subunit, referring to Domenighini etc., and Mol.Microbiol (1995)15(6): 1165-1167, the document is included in as a reference in full.

E. immunomodulator

The people's immunomodulator that is suitable as adjuvant of the present invention comprises various cell factors, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), interferon (for example interferon-γ), macrophage colony stimulatory factor and TNF.

F. bioadhesive polymer and mucoadhesive

Bioadhesive polymer and mucoadhesive also can be used as adjuvant of the present invention. Suitable bioadhesive polymer comprises the hyaluronic acid microsphere (Singh etc. of esterification, (2001) J.Cont.Rele.70:267-276) or mucoadhesive, such as the cross-linked derivant of poly-(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharide and carboxymethyl cellulose. Chitosan and derivative thereof also can be used as adjuvant of the present invention. For example, WO99/27960.

G. particulate

Particulate also can be used as adjuvant of the present invention. The particulate that is formed by biodegradable non-toxic material (such as poly-(alpha-hydroxy acid), poly hydroxybutyric acid, positive polyesters, polyanhydride, PCL etc.) and PLG (is that diameter is about 100nm-150 μ m, 200nm-30 μ m more preferably from about, the particle of 500nm-10 μ m most preferably from about) be preferred, can choose wantonly process so that its surface with negative electrical charge (as processing with SDS) or positive charge (as using cationic detergent, processing such as CTAB).

H. liposome

The example that is suitable as the Liposomal formulation of adjuvant is described in U.S. Patent No. 6,090,406, U.S. Patent No. 5,916,588 and EP 0 626 169.

I. APEO and polyoxyethylene ester formulation

Be applicable to adjuvant of the present invention and comprise polyethenoxy ether class and polyoxyethylene ester class. WO99/52549. This class preparation also comprises the polyoxyethylene sorbitan ester surfactant (WO01/21207) that mixes with Octoxinol, and the polyoxyethylene alkyl ether that mixes with at least a other non-ionic surface active agent such as Octoxinol or ester surfactant (WO01/21152).

Preferred APEO is selected from lower group: polyoxyethylene-9-bay ether (laureth 9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-bay ether, polyoxyethylene-35-bay ether and polyoxyethylene-23-bay ether.

J. polyphosphazene (PCPP)

The PCPP preparation is described in, for example, Andrianov etc., " prepare the hydrogel microsphere body by the cohesion polyphosphazene aqueous solution " (Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions), Biomaterials (1998)19(1-3): 109-115; With Payne etc., " discharging protein from polyphosphazene matrix " (Protein Release from Polyphosphazene Matrices), Adv.Drug.Delivery Review (1998)31(3)：185-196。

K. muramyl peptide

The example that is suitable as the muramyl peptide of adjuvant of the present invention comprises N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-go first muramyl-L-alanyl-D-isoglutamine (nor-MDP) and the different glutaminase acyl group-ALANINE of N-acetyl muramyl-L-alanyl-D--2-(1 '-2 '-two palmityls-sn-glycerine-3-hydroxyl phosphinylidyne oxygen)-ethamine (MTP-PE).

L. imidazo quinolone compounds

The example that is suitable as the imidazo quinolone compounds of adjuvant of the present invention comprises imiquimod and homologue thereof, be further described in Stanley, " the Mechanisms and therapy potential of imiquimod and imidazo quinolones " (Imiquimod and the imidazoquinolones:mechanism of action and therapeutic potential) Clin Exp Dermatol (2002)27(7): 571-577; And Jones, " resiquimod 3M " (Resiquimod 3M), Curr Opin Investig Drugs (2003)4(2)：214-218。

M. virion and virus-like particle (VLP)

Virion and virus-like particle (VLP) also can be used as adjuvant of the present invention. These structures contain one or more virus proteins usually, and optional and phosphatide combination or preparation. They are usually non-causes a disease and does not copy, and does not usually contain any natural viral genome. This virus protein can separate by the restructuring manufacturing or from intact virus. These virus proteins that are applicable to virion or VLP comprise the protein in following source: influenza virus (such as HA or NA), hepatitis B (such as core protein or capsid protein), HEV, measles virus, the sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, norwalk virus, HPV, HIV, the RNA bacteriophage, Q phagus beta (such as coat protein), GA bacteriophage, fr bacteriophage, AP205 bacteriophage, and Ty (such as retrotransposon Ty albumen p1). VLP also is described in in the Publication about Document: WO 03/024480, WO 03/024481, and Niikura etc., " the chimeric recombined hepatitis E hepatitis virus sample particle as potential carrier for oral vaccine delivery is presented foreign epitope " (Chimeric Recombinant Hepatitis E Virus-Like Particles as an Oral Vaccine Vehicle Presenting Foreign Epitopes), Virology (2002)293: 273-280; Lenz etc., " papillomavirus sample particle induces dendritic cells polarity to activate " (Papillomarivurs-Like Particles Induce Acute Activation of Dendritic Cells), Journal of Immunology (2001) 5246-5355; Pinto etc., " use the healthy volunteer of restructuring HPV-16L1 virus-like particle immunity to the cellullar immunologic response of HPV (HPV)-16L1 " (Cellular Immune Responses to Human Papillomavirus (HPV)-16L1Healthy Volunteers Immunized with Recombinant HPV-16 L1 Virus-Like Particles), Journal of Infectious Diseases (2003)188: 327-338; With Gerber etc., " the HPV virus-like particle is that effective oral immunity is former when jointly using with E.coli LT mutant R192G or CpG " (Human Papillomavrisu Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Entertoxin Mutant R192G or CpG), Journal of Virology (2001)75(10): 4752-4760. Virion also is described in, for example, and Gluck etc., " the in the future new technical platform of vaccine development " (New Technology Platforms in the Development of Vaccines for the Future), Vaccine (2002)20：B10-B16。

The present invention also comprises the combination of above-mentioned one or more adjuvant types. For example, following adjunvant composition can be used for the present invention:

(1) saponin(e and oil in water emulsion (WO99/11241);

(2) saponin(e (for example QS21)+nontoxic LPS derivative (for example 3dMPL) (referring to WO 94/00153);

(3) saponin(e (for example QS21)+nontoxic LPS derivative (for example 3dMPL)+cholesterol;

(4) saponin(e (for example QS21)+3dMPL+IL-12 (optional+sterol) (WO98/57659);

(5) combination (seeing european patent application 0835318,0735898 and 0761231) of 3dMPL and for example QS21 and/or oil in water emulsion;

(6) SAF contains 10% saualane, 0.4 %Tween 80,5% Pluronic block polymer L121 and thr-MDP, makes submicron emulsion or vigorous stirring formation larger particles emulsion through Micro Fluid.

(7)Ribi ^TMAdjuvant system (RAS) (Ribi Immunochem), one or more bacterial cell wall fractions that contain 2% MF59,0.2%Tween 80 and be selected from Monophosphoryl lipid A (MPL), two mycolic acid marine alga sugar esters (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox^TM); With

(8) non-toxic derivant (such as 3dPML) of one or more mineral salts (such as aluminium salt)+LPS.

Aluminium salt and MF59 are the preferred adjuvants of parenteral immunity inoculation. The bacteriotoxin mutant is preferred mucosal adjuvants.

As mentioned above, be applicable to adjuvant of the present invention can comprise following one or more:

-E.coli LT (" LT "), or its detoxification mutant are such as K63 or R72 mutant;

-cholera toxin (" CT "), or its detoxification mutant;

-the particulate that formed by biodegradable non-toxic material (such as poly-(alpha-hydroxy acid), poly hydroxybutyric acid, positive polyesters, polyanhydride, PCL etc.) (is that diameter is about 100nm-150 μ m, more preferably from about 200nm-30 μ m, the most preferably from about particle of 500nm-10 μ m);

-APEO or polyoxyethylene ester (referring to International Patent Application WO 99/52549);

-polyoxyethylene sorbitan ester the surfactant (WO01/21207) that mixes with Octoxinol, or the polyoxyethylene alkyl ether that mixes with at least a other non-ionic surface active agent such as Octoxinol or ester surfactant (WO01/21152);

-chitosan (for example International Patent Application WO 99/27960)

-immunostimulatory oligonucleotide (for example CpG ODN) and saponin(e (referring to International Patent Application WO 00/62800)

-immunostimulating double-stranded RNA.

(such as aluminium hydroxide, aluminum phosphate, Adju-Phos, hydroxy chloride aluminium oxide, aluminum orthophoshpate, aluminum sulfate etc. (for example can be referring to " vaccine design: subunit and adjuvant research " (Powell and Newman volume for-aluminium compound, Plenum Press, 1995 (ISBN 0-306-44867-X)) (hereinafter referred to as " vaccine design ") the 8th and 9 chapters, or the mixture of different aluminum compound, described compound can be taked any suitable form (such as gel, crystal, amorphous etc.), and preferably be adsorbed;

-MF59 (5% MF59,0.5%Tween 80 and 0.5%Span 85 are mixed with the submicron particle with micro-Fluidizer) (sees " vaccine design " the 10th chapter; Also can be referring to International Patent Application WO 90/14837);

-liposome (seeing " vaccine design " the 13rd and 14 chapters);

-ISCOM (seeing " vaccine design " the 23rd chapter);

-SAF contains 10% saualane, 0.4 %Tween 80,5% Pluronic block polymer L121 and thr-MDP, is changed into submicron emulsion or vigorous stirring by miniflow and forms emulsion (seeing " vaccine design " the 12nd chapter) than coarsegrain;

-RibiTM adjuvant system (RAS) (Ribi Immunochem), one or more bacterial cell wall fractions that contain 2% MF59,0.2%Tween 80 and be selected from Monophosphoryl lipid A (MPL), two mycolic acid marine alga sugar esters (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox^TM)；

-saponin adjuvant such as QuilA or QS21 (seeing " vaccine design " the 22nd chapter), also is called Stimulon^TM；

-ISCOM can not contain other detergent (WO 00/07621);

-complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA);

-cell factor is as from cytokine (IL-1 for example, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), interferon (for example interferon-γ), macrophage colony stimulatory factor, TNF etc. (seeing " vaccine design " the 27th and 28 chapters);

-monophosphoryl lipid A (MPL) or 3-O-deacylated tRNA MPL (3dMPL) (seeing " vaccine design " the 21st chapter);

The combination (european patent application 0835318,0735898 and 0761231) of-3dMPL and for example QS21 and/or oil in water emulsion;

-contain the oligonucleotides of CpG motif (referring to Krieg (2000) Vaccine, 19:618-622; Krieg (2001) Curr.Opin.Mol.Ther., 2001,3:15-24; WO 96/02555, and WO 98/16247, and WO 98/18810, and WO 98/40100, and WO 98/55495, WO 98/37919 and WO 98/52581 etc.), namely contain at least one CG dinucleotide,

-APEO or polyoxyethylene ester (International Patent Application WO 99/52549);

-polyoxyethylene sorbitan ester the surfactant (WO01/21207) that mixes with Octoxinol, or the polyoxyethylene alkyl ether that mixes with at least a other non-ionic surface active agent such as Octoxinol or ester surfactant (WO01/21152);

-immunostimulatory oligonucleotide (for example CpG ODN) and saponin(e (WO00/62800);

-immunostimulant and slaine particle (International Patent Application WO 00/23105);

-saponin(e and oil in water emulsion (WO 99/11241);

-saponin(e (for example QS21)+3dMPL+IL-12 (optional+sterol) (WO 98/57659).

Also can obtain being fit to mucous membrane or parenteral medication other (for example seeing " vaccine design: subunit and adjuvant research " (Powell and Newman compile, Plenum Press, 1995 (ISBN 0-306-44867-X)) the 7th chapter).

The mutant of LT is preferred mucosal adjuvants, " K63 " and " R72 " mutant (for example seeing International Patent Application WO 98/18928) especially, and they will strengthen immune response.

Particulate also is preferred mucosal adjuvants. Their autohemagglutinations (alpha-hydroxy acid) of preferably deriving, especially derived from PLA (" PLA "), D, the copolymer of L-lactide and glycolide or glycolic, such as poly-(D, the L-lactide-co-glycolide) (" PLG " or " PLGA "), or D, L-lactide and caprolactone copolymer. Particulate can be derived from different polymer raws, these raw materials have various molecular weight, and when being copolymer such as PLG, these raw materials have different lactides: the glycolide ratio, How to choose will be a huge selection job, and this part depends on the antigen that will use simultaneously.

SARS virus of the present invention (deactivation or attenuation), viral antigen, antibody or adjuvant can be embedded into particulate or be adsorbed to wherein. It is preferred being embedded in the PLG particulate. The PLG particulate is described in detail in Morris etc., (1994), Vaccine, 12:5-11; " mucosal vaccine " (volume such as Kiyono, Academic Press 1996 (ISBN 012410587)) the 13rd chapter, and " vaccine design: subunit and adjuvant research " (Powell and Newman volume, Plenum Press, 1995 (ISBN 0-306-44867-X)) the 16th and 18 chapters).

It is favourable that 192-GLY LT uses with the antigen of imbedding particulate, and this will significantly strengthen immune response.

Aluminium compound and MF59 are the preferred adjuvants of parenteral medication.

Described composition can comprise antibiotic.

Immunogenic composition of the present invention can be used by single dose, or as the part of therapeutic regimen. This scheme can comprise priming dose and strengthen dosage that they can be used by mucous membrane, parenteral or their various combinations.

Method of the present invention comprises that also giving the composition that animal contains effective dose antibody of the present invention treats or prevent the SARS virus relevant disease. " effective dose " of antibody of the present invention is the amount that is enough to provide for animal passive immunity protection or treatment. Preferred antibody of the present invention is specific to SARS virus antigen.

Methods for the treatment of can be combined with immunogenic composition and antibody compositions. Therefore, the present invention includes the method for the treatment of or prevention SARS virus relevant disease, described method comprises the specific antibody of using the immunogenic composition that contains immune effective dose SARS virus antigen and using the SARS virus antigen of effective dose. Immunogenic composition and antibody can be used or use separately simultaneously. The present invention also comprises a kind of composition, wherein comprises the immunogenic composition that contains immune effective dose SARS viral antigen, and contains the specific antibody of the SARS virus antigen of effective dose

SARS virus antigen of the present invention and antibody can the polynucleotides form be used. Then SARS virus antigen and/or antibody protein express in vivo.

SARS virus antigen of the present invention and antibody can be carried with one or more genophores, adopt the standard gene carrying method to inoculate to give by nucleic acid immunization. It is known in the art carrying the method for gene. Referring to for example United States Patent(USP) Nos. 5,399,346,5,580,859,5,589,466. Described construction can by in subcutaneous, epidermis, the corium, in the muscle, in the intravenous, mucous membrane (such as nose, rectum and vagina), peritonaeum, oral route or its make up to carry (for example injection). Yang etc. [(2004) Nature 428:561-564] described the 0th, 3 and 6 weeks to mouse muscle in the DNA (preparing with 200 μ l PBS pH 7.4) of injection 25 μ g coding furcella antigen.

An example that can be used for the replication defective gene delivery carrier of the present invention's practice is any α viral vectors, is described in for example United States Patent(USP) Nos. 6,342,372; 6,329,201 and WO 01/92552.

Many systems based on virus that gene are transported to mammalian cell have been developed. For example, retrovirus is a convenient platform of gene delivery system. Can and be packaged into retrovirus with technology known in the art with selected sequence insertion vector. Then the separating heavy papova is also incited somebody to action in vivo or the external experimenter's cell that is transported to. Many retrovirus system (U.S. Patent No. 5,219,740 had been described; Miller ﹠ Rosman, BioTechniques (1989) 7:980-990; Miller, A.D., Human Gene Therapy (1990) 1:5-14; Scarpa etc., Virology (1991) 180:849-852; Burns etc., Proc.Natl.Acad.Sci.USA (1993) 90:8033-8037; With Boris-Lawrie ﹠ Temin, Cur.Opin.Genet.Develop. (1993) 3:102-109.

Many adenovirus vectors also are described. Different from the retrovirus that is integrated into host genome, adenovirus is retained in outside the chromosome, therefore can make the least risk relevant with inserting mutagenesis (Haj-Ahmad and Graham, J.Virol. (1986) 57:267-274; Bett etc., J.Virol. (1993) 67:5911-5921; Mittereder etc., Human Gene Therapy (1994)5: 717-729; Seth etc., J.Virol. (1994) 68:933-940; Barr etc., Gene Therapy (1994) 1:51-58; Berkner, K.L.BioTechniques (1988) 6:616-629; With Rich etc., Human Gene Therapy (1993) 4:461-476). The codon optimization model that the research adenovirus is carried the gene of the structural antigen furcella of encoding SARS coronavirus S1, memebrane protein and nucleocapsid protein in macaque, and find that this will cause strong Neutralizing antibody response (Gao etc., (2003) Lancet 362 (9399): 1895-1896).

In addition, various adeno-associated virus (AAV) carrier system also can be used for the gene conveying. Be easy to make up the AAV carrier with technology well known in the art. Referring to for example United States Patent(USP) Nos. 5,173,414 and 5,139,941; WO 92/01070 (on January 23rd, 1992 is open) and WO 93/03769 (on March 4th, 1993 is open); Lebkowski etc., Molec.Cell.Biol. (1988) 8:3988-3996; Vincent etc., Vaccines90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B.J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N.Current Topics in Microbiol.and Immunol. (1992) 158:97-129; Kotin, R.M.Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; With Zhou etc., J.Exp.Med. (1994) 179:1867-1875.

Other carrier system that is used for by mucous membrane or other approach delivery of polynucleotide is the recombinant poxvirus vaccine that enteron aisle is used, be described in little Small, (the U.S. Patent No. 5 such as P.A., 676,950, on October 14th, 1997 delivered, and included this paper in as a reference) and vaccinia virus and fowlpox virus. For example, the vaccinia virus recombinant of expressing said gene can make up by the following method. At first the DNA of encoding SARS antigen or antibody or antibody coding sequence are inserted and make it adjoin the cowpox promoter in the suitable carrier and be positioned at the cowpox dna sequence dna, such as the flank of coding thymidine kinase (TK) sequence. Then the cell that comes transfection to infect with cowpox simultaneously with this carrier. Can be inserted in this viral genome by the gene of homologous recombination with cowpox promoter and coding sequence interested. Can be when having 5-bromine deoxyguanosine cultured cell and select wherein resistance virus and have a liking for bacterial plaque and select gained TK-recombinant.

Perhaps also available fowlpox virus (such as chicken pox and canary pox virus) is carried the gene of code book invention SARS virus antigen or antibody. The known immunogenic recombinant fowlpox virus of mammal pathogen of will expressing is applied to non-avian species protective immunity can be provided. Because fowl pox only copies in the susceptible bird productively so can mammalian cell-infecting, in human and other mammal, adopt the fowl pox carrier desirable especially. The method of making recombinant fowlpox virus is known in the art, can make with inherited recombinant according to the method for above-mentioned manufacturing vaccinia virus. Referring to, for example, WO 91/12882; WO 89/03429; With WO 92/03545. The carrier that also can use picornavirus to derive. (referring to, for example, United States Patent(USP) Nos. 5,614,413 and 6,063,384).

The molecule coupling carrier also can be used for gene such as [Proc.Natl.Acad.Sci.USA (1992) 89:6099-6103] described adenovirus chimeric vectors such as Michael etc. [J.Biol.Chem. (1993) 268:6866-6869] and Wagner and carries.

Can adopt easily cowpox infection/transfection system that the inducibility transient expression (for example, SARS virus antigen or antibody expression box) of coded sequence interested is provided in host cell. In this system, at first use vaccinia virus recombinant's Infection in Vitro cell of coding T7 phage rna polymerase. This polymerase shows cleverly specificity, and namely it only transcribes the template with the T7 promoter. After the infection, interested polynucleotides are transfectional cell under the T7 promoters driven. That expresses in kytoplasm is transcribed into RNA from the polymerase of vaccinia virus recombinant with the DNA of transfection, then by the translating mechanism of host cell RNA is translated into protein. The method can be made a large amount of RNA and translation product thereof in high-level ground momently in kytoplasm. Referring to, for example, Elroy-Stein and Moss, Proc.Natl.Acad. Sci.USA (1990) 87:6743-6747; Fuerst etc., Proc.Natl.Acad.Sci.USA (1986) 83:8122-8126.

As infecting with cowpox or recombinant of fowlpox virus, or with the alternative that other viral vectors is carried gene, amplification system can be used, high-caliber expression will be obtained after being introduced into host cell. Specifically, but engineering makes up the t7 rna polymerase promoter be positioned at before the t7 rna polymerase code area. Translation will produce t7 rna polymerase from the RNA of this template, and then transcribe out more template. Produce simultaneously the cDNA that its expression is subjected to the control of T7 promoter. Therefore, some will cause transcribing of required gene by the t7 rna polymerase of translating this amplification template RNA and producing. Because some t7 rna polymerases are essential by this amplification of startup, can the T7 RNA polymerase be introduced cell to cause responsive transcription with template. Polymerase can be introduced as protein or at the plasmid of coding RNA polymerase. About T7 system and their application in transformant can referring to, for example, WO 94/26911; Studier and Moffatt, J.Mol.Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao etc., Biochem.Biophys.Res.Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993) 21:2867-2872; Chen etc., Nuc.Acids Res. (1994) 22:2114-2120; With U.S. Patent No. 5,135,855.

Immunogenic composition of the present invention also can contain diluent, such as water, salt solution, glycerine, alcohol etc. In addition, also can contain auxiliary substance in the described immunogenic composition, such as wetting agent or emulsifying agent, pH buffer substance etc.

Be used for immunogenic composition of the present invention and can be applied to animal. The animal that is fit to the inventive method comprises people and other primate, comprises non-human primates, such as chimpanzee, and other apes and monkey class; Farm-animals is such as ox, sheep, pig, goat and horse; Domestic animal is such as dog and cat; Experimental animal comprises rodent, such as mouse, rat and cavy; Birds comprise domestic birds, wild birds and game birds, such as chicken, turkey and quail bird, duck, goose etc. Being applicable to animal of the present invention can be any age, comprises adults and new born animal. Transgenic animals also can be used for the present invention.

Immunogenic composition of the present invention can be used to treatment or prevention SARS virus relevant disease.

Composition of the present invention be preferably pharmaceutically acceptable or pharmacology on acceptable. Specifically, it not is to be that biology or other side are unwanted that described composition is preferably, namely this material can preparation or the form of composition be applied to individuality and can not cause any undesirable biological effect, or with harmful mode and the interaction of its contained composition component.

Pharmaceutically acceptable salt can be used for composition of the present invention, for example, and mineral salt (example hydrochloric acid salt, hydrobromate, sulfate or sulfate) and organic acid salt (such as acetate, propionate, malonate or benzoate). Useful especially protein material is seralbumin, key hole _ hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin and is proficient in the other oroteins known by the technical staff in this field. Composition of the present invention also can contain liquid or excipient, such as water, salt solution, glycerine, glucose, alcohol etc. (they can individualism or combination exist), and the material of wetting agent, emulsifying agent or pH buffer and so on. Also available liposome is as the carrier of the present composition.

Vaccine of the present invention is made and detected to useful SARS specific reagent or analysis determination method. This analysis determination method for example comprises: 1) titration of virus and have a liking for plaque assay to quantize infectious virus particle, 2) with constant virus and the variable serum dilution mensuration that neutralizes, 3) two one step RT-PCR systems (Light Cycler-Roche) are to detect negative strand viruses RNA, make target sequence be positioned at the N gene, possible hypersensitivity is provided, with 4) ELISA and western engram analysis, to detect and purified virus protein.

In addition, can generate the rabbit polyclonal antiserum with the antibody reagent that obtains anti-SARS-CoV (and confirm in and the inducing of antibody). The sampling plan that generates this reagent is as follows. At first in suitable cultured cell (such as the Vero cell), cultivate virus, and by 20% sucrose (w/v) pad precipitation. Then sediment is processed to be further purified with glycerine-potassium tartrate gradient. Then will contain virulent part dilution and pass through Ultracentrifugation. Then sediment is dissolved in PBS and uses C₃H ₄O ₂(beta-propiolactone, BPL) inactivation of viruses. The the 0th, 14 and 28 day with adjuvant IFA mix 1 * 10⁹Two rabbits of inactivated virus particle subcutaneous (SC) immunity. Gather rabbit blood in the 0th (before the inoculation), 13,28 and 35 (after 1 week of for the third time immunity) sky. Detect the anti-SARS-CoV albumino reaction of this scheme gained serum by the western trace, found that with major structural protein furcella (S), film (M) and nucleocapsid (N) albumen to react.

J. the coronavirus vaccine that is just occurring

The popular people of causing of SARS understand the virus infections that is caused by coronavirus to be increased. Vaccine of the present invention can be used for preventing or treating the coronavirus strain that will occur, and comprises the SARS virus that will occur.

The invention provides a kind of vaccine, wherein comprise subunit's preparation of one or more human corona virus's antigens of (or killing) human corona virus, attenuation human corona virus, cracking human corona virus goods or restructuring or the purifying of deactivation, wherein said human corona virus is not sars coronavirus. Randomly, described human corona virus is not the 229E coronavirus. Randomly, described human corona virus is the OC43 coronavirus. Randomly, described human corona virus is not the NL63 coronavirus. Therefore, the invention provides vaccine as hereinbefore defined, wherein, described human corona virus is not sars coronavirus, is not the 229E coronavirus, is not the OC43 coronavirus, and is not the NL63 coronavirus. This vaccine can be used for preventing and/or treating the human corona virus who will occur to be infected.

The present invention also provides the vaccine that contains following component: (a) (or killing) human corona virus of deactivation, the attenuation human corona virus, cracking human corona virus goods, or the subunit of one or more human corona virus's antigens of restructuring or purifying, wherein said human corona virus is not sars coronavirus, definition as mentioned; (b) (or killing) human corona virus of deactivation, the attenuation human corona virus, cracking human corona virus goods, or subunit's preparation of one or more human corona virus's antigens of restructuring or purifying, wherein said human corona virus is sars coronavirus. This vaccine can be used for preventing and/or treating SARS and other human corona virus.

Except the vaccine that contains from the antigen of more than one coronavirus types is provided, the present invention also provides a kind of vaccine, wherein contain the antigen from the multiple strain of coronavirus of the same race, such as the antigen of the different strains of the different strains of sars coronavirus or the coronavirus outside the SARS coronavirus. In one embodiment, described vaccine comprises at least from two kinds of coronavirus strains, or the antigen of at least three kinds of coronavirus strains. In one embodiment, described vaccine comprises the antigen from least two kinds of coronavirus types. In one embodiment, described vaccine comprises various known coronavirus types (I type, II type and III type) at least a antigen approximately. This vaccine has the pattern of present influenza vaccines.

Selection to the coronavirus that is used for vaccine of the present invention and/or coronavirus can be based on various standards. For example, selection can be based on the virus and/or the strain that the geographic area (such as the Northern Hemisphere or the Southern Hemisphere, concrete country etc.) of throwing in had been detected at vaccine. Selection can be supervised based on animal (depending on the virus that for example detects) result in the patient because of the respiratory tract infection hospitalization. Can select every year, for example before entering the winter. Also can annual vaccine inoculation, equally also with the patterns of influenza vaccines.

Preferred vaccine should have sufficient immunogenicity so that the neutrality immune response to be provided, and more preferably provides protectiveness and/or therapeutic immunization to reply. Particularly preferred vaccine should meet the effectiveness requirement of when the time comes appointment of WHO.

The contained preferred subunit antigen of vaccine of the present invention is the spike protein of purifying, more preferably with oligomerization (for example trimerization) form. Spike protein can be cut into its S1 and S2 product, perhaps is not cut.

The technology that above-mentioned selection virus and/or strain are made vaccine also can be used to select the HR1 of obtainable suitable virus and/or strain and HR2 sequence so that above-mentioned therapeutic peptide to be provided.

III. diagnosis composition of the present invention and method

The invention provides the method that detects sars coronavirus. Detect patient's sample and can be used for the diagnosis and detection virus infections. Detect donated blood and can be used for preventing that the careless virus that occurs in the blood transfusion process from propagating. Detection method mainly comprises three aspects: detect SARS virus nucleic acid; Detect SARS virus albumen; With the immune response that detects anti--SARS virus. The invention provides all these three kinds of methods.

When this paper mentions the concrete oligonucleotide probe of nucleotide sequence and primer, " similarly " sequence comprises the sequence that those are identical with known SARSV genome sequence at least 90%, and is included on probe or the primer length and SARSV genome sequence at least 95% identical, at least 99% identical and 100% identical sequence.

Term " target nucleic acid zone " or " target nucleic acid " represent the nucleic acid molecules with " target sequence " that will be amplified here. Target nucleic acid can be strand or two strands, and can comprise other sequence that is not amplified beyond the target sequence. The specific nucleotide sequence of the target nucleic acid that term " target sequence " expression will be amplified. Target sequence can comprise and be contained in the target molecule and zone Probe Hybridization, under appropraite condition probe will with this regional stability hybridization. " target sequence " also can comprise oligonucleotide binding primer and the multiplexed sequence that extends as template with target sequence. Described target nucleic acid can be strand originally, term " target sequence " also refer to target nucleic acid in " target sequence " that exist complementary sequence. If " target nucleic acid " is double-stranded originally, then term " target sequence " represents just (+) chain and negative (-) chain simultaneously.

Term " primer " or " Oligonucleolide primers " here represent when placing under the condition of inducing the synthetic primer extension products-oligonucleotides that namely has nucleotides and polymerisation induced agent (such as DNA or RNA polymerase) and have suitable temperature, pH, metal concentration and salinity-initiations complementary dna chain to synthesize. Primer be preferably strand increasing most effectively, but also can be double-stranded. If double-stranded, at first to process to separate to primer its two chains, then be used for preparing extension products. Denaturing step is finished by heating usually, but also available bases is processed, and then neutralization. Therefore, " primer " is complementary with template, and compound obtaining primer/stamp complex by hydrogen bond or hybridization and template, thus under the effect of polymerase, cause syntheticly, in the DNA building-up process, extend primer by adding the covalently bound base with the template complementation at primer 3 ' end.

Refer to term " probe " or " oligonucleotide probe " structure that consisted of by above-mentioned polynucleotides, described polynucleotides contain with the target nucleic acid analyte in the nucleotide sequence of the nucleic acid array complementation that exists. The polynucleotide region of probe can be made of DNA and/or RNA and/or synthetic nucleoside analog. When " oligonucleotide probe " is used for 5 ' nuclease mensuration, such as TaqMan^TMTechnology, probe will contain the quencher thing of at least a fluorescer and at least a 5 ' the endonuclease enzymatic activity digestion that is used to this reaction to detect the target oligonucleotide sequence of any amplification. In this article, described oligonucleotide probe is adjoining its 5 ' end and will contain the di-phosphate ester tendon of sufficient amount, and used like this 5 ' to 3 ' nuclease can effectively be degraded the probe of combination to separate fluorescer and quencher thing. When oligonucleotide probe was used to the TRA technology, it will be by suitable mark, and was as described below.

Hybridization sequences needn't have the good complementarity that stable hybridization is provided. In most cases, ignore four or the ring that forms of polynucleotides more, when base mismatch is less than about 10%, just can form stable crossbred. Therefore, term " complementation " here is illustrated under the experimental condition and forms the oligonucleotides of stablizing duplex with its " complement " and usually have an appointment 90% or higher homology.

Term " hybridization " and " hybridization forms " to be illustrated in abundant complementation and to form by the Watson-Crick base pairing between the nucleotide sequence of complex and form complex. When primer and target (template) " hybridization ", this species complex (or crossbred) is enough stable, plays the function that for example causes the synthetic required primer of archaeal dna polymerase of DNA.

Stringent hybridization condition generally includes following requirement: salinity is lower than about 1M, more generally is lower than about 500mM, preferably is lower than about 200mM; Hybridization temperature can be low to moderate 5 ℃, but usually above 22 ℃, more generally is higher than about 30 ℃, preferably is higher than about 37 ℃. Long fragment may need higher hybridization temperature with specific hybrid. The other factors of impact hybridization stringency comprises base composition and the length of complementary strand, whether has the degree of organic solvent and base mispairing, and the combination of institute's operation parameter is more important than the absolute figure of any single parameter. Controllable other hybridization conditions comprises buffer type and concentration, pH value of solution, whether has closed reagent or closure protein solution and their concentration, detergent kind and the concentration that reduces background combination (such as repetitive sequence), polymer class molecule, metal ion and concentration thereof, chelating agent and the concentration thereof that can improve the polynucleotides relative concentration, and other condition known in the art. Can use stringency lower and/or more meet the hybridization conditions of physiological requirement, measure in (molecular beacon assay) process at the real time measure such as the molecular labeling of pcr amplification monitoring this moment, and the polynucleotide amplification cycling switch substrate of mark is combined with complementary probe polynucleotides. The hybridization conditions that this stringency is lower also comprises the other side of the method for example reverse transcription or the effective solution condition of PCR.

" biological sample " refers to separate tissue, cell or the humoral sample from object here, wherein generally includes the antibody of being made by this object. Typical sample includes but not limited to blood, blood plasma, serum, excreta, urine, marrow, bile, spinal fluid, lymph liquid, skin samples, skin, respiratory tract, enteron aisle and genitourinary tract, tears, saliva, phlegm, mucous membrane, milk, haemocyte, organ, tissue, living tissue (for example lung, liver, kidney), and vitro cell culture sample, comprising but be not limited to cell and be organized in the conditioned medium that growth obtains in the culture medium, for example recombinant cell and cellular component. Other sample also can be used for diagnosis, comprises fecal specimens and nasopharynx aspirate.

Term " antibody " comprises polyclone and monoclonal antibody goods, and comprises hybrid antibody, variation antibody, chimeric antibody and humanized antibody, and hybridization (chimeric) antibody molecule (referring to, for example, Winter etc., (1991) Nature349: 293-299; With United States Patent (USP) 4,816,567); F (ab ')₂And F (ab) fragment; The Fv molecule (non-covalent heterodimer, referring to, for example, Inbar etc., (1972) Proc Natl Acad Sci USA69: 2659-2662; With Ehrlich etc., (1980) Biochem19: 4091-4096); ScFv molecule (sFv) (referring to, for example, Huston etc., (1988) Proc Natl Acad Sci USA85: 5879-5883); Oligomer; Dimerization and trimerization antibody fragment construction; Microbody (referring to, for example, Pack etc., (1992) Biochem31: 1579-1584; Cumber etc., (1992) J Immunology149B: 120-126); Humanized antibody molecules (referring to, for example, Riechmann etc., (1988) Nature332: 323-327; Verhoeyan etc., (1988) Science239: 1534-1536; Open on September 21st, 2,276,169,1994 with the open No.GB of BP); And available from any functional fragment of this molecule, wherein said fragment keeps the specific binding characteristic of parental antibody molecule.

Term " monoclonal antibody " refers to contain the antibody compositions of a group homogeneous antibody here. This term is not subjected to the restriction in antibody type or source, and is not subjected to the restriction of its preparation method. This term comprises all immunoglobulin (Ig)s.

The method for preparing polyclone and monoclonal antibody is known in the art. Polyclonal antibody is by producing with the suitable animal of interested antigen immune (such as mouse, rat, rabbit, sheep or goat). For strengthening immunogenicity, before the immunity antibody is connected with carrier. The normally large metabolism of suitable carrier is large molecule slowly, such as amino acid, amino acid copolymer, lipid concentration thing (such as oil droplet or liposome) and the inactivated virus particle of protein, polysaccharide, PLA, polyglycolic acid, polymerization. This carrier is be proficient in this field known by the technical staff. In addition, antigen can be conjugated on the bacterial toxoid, such as diphtheria, lockjaw, cholera toxin etc., to strengthen its immunogenicity.

When needs large volume serum, with rabbit, that sheep and goat prepares polyclonal antiserum is better. Simultaneously because can obtain mark anti--rabbit, anti--sheep and anti--goat antibody, so these animals are preferably design alternatives. Immunity is carried out usually like this: antigen is mixed or emulsification with salt solution (better with adjuvant such as Freund's complete adjuvant (" FCA ")), and then parenteral (normally subcutaneous or intramuscular) is injected this mixture or emulsion. The antigen of using afterwards salt solution (preferably using incomplete Freunds adjuvant (" FIA ")) to join in 2-6 week is injected one or many with reinforced immunological. Can carry out external immunity with methods known in the art in addition and produce antibody. Then the animal from immunity obtains polyclonal antiserum.

As mentioned above, usually improve one's methods to make monoclonal antibody according to the method [(1975) Nature 256:495-497] of Kohler and Milstein or its.

Nucleic acid detection method

The method of known many amplified target sequences is such as PCR (PCR), reverse transcription PCR (RT-PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and based on the amplification (NASBA) of nucleotide sequence, the amplification (TMA) of transcriptive intermediate. These methods are generally described in below with reference in the document: (PCR) United States Patent (USP) 4,683, and 195,4,683,202 and 4,800,159; (RT-PCR) United States Patent (USP) 5,310, and 652,5,322,770; (LCR) EP application No., 320,308 (on June 14th, 1989 is open); (SDA) United States Patent(USP) Nos. 5,270,184 and 5,455,166, and G.T.Walker " practical application of strand displacement amplification " (Empirical Aspects of Strand Displacement Amplification), be selected from " PCR method and application " (PCR Methods and Applications), 3 (1): 1-6 (1993), Cold Spring Harbor Laboratory Press; (TMA) U.S. Patent No. 5,399, and 491; (NASBA) L.Malek etc. is " based on the amplification (NASBA of nucleotide sequence^TM) ", be selected from " molecular biology method " (Methods in Molecular Biology), the 28th volume 36 chapters: by the method for nonradioactive probe analysis of nucleic acids, 1994 compile P.G.Isaac, Humana Press, Inc., Totowa, N.J.. PCR method can comprise that some change to quantize target sequence, for example by PCR in real time analysis (especially such as United States Patent (USP) 5,210,015,5,487,972,5,994,056,6,171,785 is described). (above-mentioned reference is included in this paper as a reference in full).

Whether one of the present invention be used for existing the embodiment of SARS virus may further comprise the steps in the test sample: provide and suspect the sample that contains the SARS virus nucleic acid target; Use Oligonucleolide primers described here, especially contain those primers of kit described here, increasing with any known nucleic acid amplification technologies (comprising any technology of mentioning here) is contained in template sequence in the described SARS virus nucleic acid target; And the template sequence that detects amplification, wherein exist the template sequence that increases to illustrate in the described sample and have SARS virus.

Amplification technique generally includes and uses two kinds of primers. When target sequence was strand, this technology generally includes made complementary strand to obtain the preliminary step of double-stranded target. Two kinds of primers are hybridized from the different chains of double-stranded target, and then extend. Extension products can be used as target with further hybridization/extension. Clean effect is the template sequence in the amplified target, and 5 ' and 3 ' end of template can be by the location positioning of two primers in the target. Perhaps, if one of primer or both are contained promoter sequence, (by transcribing) target (as TMA) then can increase with RNA polymerase.

The invention provides amplification and/or detect template in the SARSV viral nucleic acid or method and the kit of target sequence. The invention provides the kit of the primer of contained template sequence in the SARSV nucleic acid target that contains to increase, described kit comprises the first primer and the second primer, wherein said the first primer contains and the basic complementary sequence of the part of described template sequence, and described the second primer contains and the basic complementary sequence of the part of the complement of described template sequence, wherein, basic complementary sequence has been determined two ends of the template sequence that will be amplified in the described primer. Kit of the present invention also is equipped with and template sequence and/or the basic complementary probe that also can hybridize with it of its complement. This probe can be used for hybridization technique with the template of detection amplification, or is used for separating the template of (namely catching) amplification or initial target nucleic acid.

Target primer and/or probe in kit of the present invention also is equipped with to produce and detects are to help measures of quantization result (for example, Fille etc., 1997 Biotechniques 23:34-36).

Kit of the present invention also is equipped with archaeal dna polymerase, and it is the heat endurance archaeal dna polymerase normally, and will use the non-isothermal amplification procedure this moment. This kit also can comprise provides dNTP, magnesium salts (MgCl for example₂), buffer solution etc.

Kit of the present invention also comprises a pair of above primer (for example be used for nested amplification), when a kind of primer than an above primer to referring to. Described kit also can contain a plurality of probes.

Oligomerization probe and primer

In conjunction with the nucleic acid detection method of the invention described above, available SARSV genome has sequence similarity or complementary oligomer. Here the SARSV genome sequence of mentioning produces probe and primer, in the test for detection of the nucleic acid in the test specimen. Can come designing probe according to the conservative nucleotides zone of polynucleotides interested or the non-conservation nucleotides zone of polynucleotides interested. It is be proficient in this field known to the skilled that design can make the method for measuring optimized probe. Usually, when high specific of needs, can produce nucleic acid probe according to non-conservation zone or unique zone, when to (such as) the multigene family different members or such as relevant animals such as mouse and people in the closest nucleotides zone of relation when detecting, can produce nucleic acid probe according to the conservative zone.

Can be as shown here according to SARSV genome and/or the preferred genomic conserved region of SARSV, and/or primer especially described here and probe sequence, can prepare and contain about 8 or the oligomer of polynucleotides more, this oligomer can be hybridized with normal chain or its complement of SARSV RNA, and hybridizes with SARSV cDNA. These oligomers can be used as the probe that detection contains (comprise and separating and/or mark) polynucleotides of SARSV nucleotide sequence, and/or as the primer of transcribing and/or copy target SARSV sequence. Described oligomer contains the target polynucleotide sequence, and this sequence is made of the nucleotides with the complementation of target SARSV nucleotide sequence; This sequence long enough also can form the required sufficiently stable duplex of this purpose with the complementation of SARSV sequence. For example, if purpose is the analyte that contains target SARSV sequence by fixedly separating, described oligomer can contain long enough and with the enough complementary polynucleotide region of target SARSV sequence, fully stable with the duplex in conjunction with obtaining analyte to be fixed on the surface of solids by analyte and oligomer under separation condition. Equally, for example, if this oligomer is to transcribe and/or copy target SARSV sequence in the analyte polynucleotides as primer, then will contain the fully complementary polynucleotide region of long enough and target SARSV sequence in this oligomer, under polymerizing condition, to make polymerizer from the primer continuous replication of the stable duplex form that contains this target sequence. Also for example, if this oligomer is used as label probe or will be combined with polymer, the target polynucleotide region is with long enough and sufficient complementarity is arranged to form stable heteroduplex body structure can detect this duplex with label probe and/or polymer. Described oligomer is minimum to contain 4 continuous nucleotides with the complementation of target SARSV sequence of having an appointment; Usually, described oligomer contains at least about 8 continuous nucleotides with the complementation of target SARSV sequence, preferably contain with the complementation of target SARSV sequence at least about 14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 continuous nucleotides are up to 50,75,100,200 continuous nucleotides or more of having an appointment.

Usually, in the method (for example PCR, RT-PCR, TMA) based on amplification, oligomer will be used as primer sets, the more conservative part (in coronavirus) of a member in this primer sets and SARSV genome has sequence similarity or complementarity, and other member of this primer sets has sequence similarity or complementarity with very not conservative part. The mode amplified target zone that this primer sets can be used to know with this field. Usually, 5 ' non-translational region (5 ' UTR) and 3 ' non-translational region (3 ' UTR) are the most conservative zones. Fig. 8 has shown the contrast of several coronavirus 5 ' UTR. Figure 10 has shown the contrast of several coronavirus 3 ' UTR. Fig. 9 and 11 show respectively to increase sequence of 5 ' UTR and the preferred primer of 3 ' UTR. Easily design other primer and probe based on the sequence contrast that provides here.

Yet described oligomer does not need only to be made of the sequence with the complementation of target SARSV sequence. It also contains nucleotide sequence (for example promoter) or the other parts that are fit to this oligomer application target in addition. For example, if this oligomer is used through for example primer of pcr amplification SARSV sequence, they can contain the sequence that forms restriction enzyme site when for duplex form, and this site is conducive to the sequence of particle amplification. Again for example, if this oligomer will be used as hybridization assays " capture probe ", they also will comprise and the binding partners that contains with the oligomer coupling of the oligonucleotide sequence of target SARSV sequence complementation. Described oligomer can comprise or be coupled to molecule or the sequence of other useful type known in the art, and is suitable for various purposes, comprises the labeled nucleotide probe.

Table 4 (SEQ ID NOS:1021-6020) has shown forward and the anti-phase primer of the effective amplification SARSV nucleic acid that is used for diagnosis and screening technique.

Be diagnosis and screening, the primer and the probe that preferably detect SARS nucleic acid are SEQ ID NOS:7332-7336 (forward primer), SEQ ID NOS:7337-7341 (reverse primer) and SEQ ID NOS:7342-7352 (probe). These primers and probe can effectively detect 3 ' UTR sequence.

Above-mentioned any forward primer can with above-mentioned any reverse primer SARSV nucleic acid that is used in combination to increase. Amplified production can be with above-mentioned any probe in detecting (or catching). The combination that particularly preferred forward and reverse primer and being used for detects the probe of amplified production is: forward SEQ ID NO:7332, reverse SEQ ID NO:7337,7338,7339 or 7341, probe SEQ ID NO:7342; Forward SEQ ID NO:7333 or 7334, oppositely arbitrary as probe among SEQ ID NO:7340 and the SEQ ID NO:7343-7351; Forward SEQ ID NO:7335, reverse SEQ ID NO:7340 or 7341, probe can be among the SEQ ID NO:7342-7352 arbitrary. Other combination of forward and reverse primer and proper probes is that to be proficient in the technical staff in this field not doubt by top information.

Other primer and the probe that preferably detects SARS nucleic acid that is used for diagnosis and screening is SEQ ID NOS:7353-7362 (forward primer), SEQ ID NOS:7363-7373 (reverse primer) and SEQ ID NOS:7374-7385 (probe). These primers and probe can effectively detect the sequence among the 5 ' UTR.

The use capable of being combined of above-mentioned primer is with amplification SARSV nucleic acid, make up as follows: forward primer is arbitrary among the SEQ ID NO:7353-7356, reverse primer is arbitrary among the SEQ ID NO:7363-7366,7368, and amplified production detects (or catching) with probe SEQ ID NO:7374; Forward primer is arbitrary among the SEQ ID NO:7357-7362, and reverse primer is arbitrary among SEQ ID NO:7367, the 7369-7373, and amplified production detects (or catching) with probe SEQ ID NO:7375-7385. The combination of particularly preferred forward and reverse primer and probe is: forward primer is SEQ ID NO:7353-7356, and reverse primer is arbitrary among the SEQ ID NO:7363-7366, and probe is SEQ ID NO:7374; Forward primer is SEQ ID NO:7357-7358, and reverse primer is SEQ ID NO:7367,7369, and probe is SEQ ID NO:7375 or 7376; Forward primer is SEQ ID NO:7357-7359, and reverse primer is SEQ ID NO:7367,7369 or 7370, and probe is SEQ ID NO:7375 or 7376. Preferred combination is SEQ ID NO:7353 or 7354 and SEQ ID NO:7363 or 7364, and probe is SEQ ID NO:7374. Other combination of forward and reverse primer and proper probes is that to be proficient in the technical staff in this field not doubt by top information. In the 3 ' UTR of SARS and some other coronavirus (since about 70-80 base of 3 ' end), there is the sequence (SEQ ID NO:7386) of eight nucleotides guarding especially particularly useful when identifying SARSV. Contain that this regional primer should to have more the reverse primer of specific sequence area combined with SARS.

Except mentioned above, in SARSV, identified the distinctive intergenic sequence of coronavirus (IS) (seeing above). IS comprises sequence A CGAAC (SEQ ID NO:7293) at least, and this sequence appears at each ORF of viral genome (ORF) upstream. 5 ' the UTR that comprises IS at 5 ' the terminal or position of adjoining IS of each viral mRNA by montage. Therefore, the primer that contains IS or its complement can be used to amplicon virus nucleic acid, comprises the cDNA that makes from viral RNA. Therefore, the present invention includes one group of primer, one of them primer comprises ACGAAC (SEQ ID NO:7293) or its complement (SEQ ID NO:7387), and a primer comprises from the genomic any proper sequence of SARS or complementary series. Be used for detecting and/or catch viral RNA or can comprise the IS sequence from the useful probe of the cDNA of viral RNA manufacturing, or its complement, as mentioned above.

The primer of one group of SARS sequence (especially passing through RT-PCR) that is used for increasing adopts SEQ ID NOs 6562,6563,6564 and 6565. Certainly, 6562 is 6564 to be sense primers, and 6563 and 6565 is antisense primers. Primer SEQ ID NOS:6562 and 6565 can be used for for the first time amplification, and primer SEQ ID NOS:6563 and 6564 have been adopted in for the second time nested amplification. In some embodiments of the present invention, these four kinds of primers all are left out.

A kind of kit that is used for amplification and detects SARS sequence (especially passing through RT-PCR) adopts SEQ ID NOs 6567 and 6568 as primer, and SEQ ID NO 6566 comes extension increasing sequence as probe (especially using TAMRA for example and/or FAM mark). In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit that is used for amplification and detects SARS sequence (especially passing through RT-PCR) adopts SEQ ID NOs 7395 and 6568 as primer, and SEQ ID NO 6566 comes extension increasing sequence as probe (especially using TAMRA for example and/or FAM mark). In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit of the SARS sequence (especially Nucleoprotein Gene) that is used for increasing adopts SEQ ID NOs 6560 and 6561 as primer. In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit of the SARS sequence that is used for increasing adopts SEQ ID NOs 6496,6497,6562,6563,6564 and 6565 as primer. In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit of the SARS sequence that is used for increasing adopts SEQ ID NOs 6562,6563,6564 and 6565 as primer. In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit of the SARS sequence that is used for increasing adopts SEQ ID NOs 6500,6501,6502 and 6503 as primer. In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit of the SARS sequence that is used for increasing adopts SEQ ID NOs 6496,6497,6500,6501,6502,6503,6562,6563,6564 and 6565 as primer. In some embodiments of the present invention, these primers and probe all are left out.

A kind ofly be used for amplification and detect the SARS sequence (especially by PCR in real time, for example TaqMan^TM) kit adopt SEQ ID NOs 6567 and 6568 as primer, SEQ ID NO 6566 comes extension increasing sequence as probe (especially using TAMRA for example and/or FAM mark). In some embodiments of the present invention, these primers and probe all are left out.

A kind ofly be used for amplification and detect the SARS sequence (especially by PCR in real time, for example TaqMan^TM) kit adopt SEQ ID NOs 7395 and 6568 as primer, SEQ ID NO 6566 comes extension increasing sequence as probe (especially using TAMRA for example and/or FAM mark). In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit that is used for increasing and detects the SARS sequence adopts SEQ ID NOs 6562,6565 and 6568 as primer, and SEQ ID NO 7396 and 7397 comes extension increasing sequence as probe (especially using TAMRA for example and/or FAM mark). In some embodiments of the present invention, these primers and probe all are left out.

A kind of kit that is used for increasing and detects the SARS sequence is with containing the oligonucleotides of SEQ ID NO:9780 as forward primer, with the oligonucleotides that contains SEQ ID NO:9781 as reverse primer, and with the oligonucleotides that contains SEQ ID NO:9782 as probe.

The sequence that the preferred RT-PCR of being used for and LightCycler analyze comprises SEQ ID NOs 6562,6568,6565,7396 and 7397. In some embodiments of the present invention, these primers and probe all are left out.

By the method for this oligomer of preparation known in the art, comprising for example comprising excision, transcribing or chemical synthesis process. The target polynucleotides of selecting oligomer according to purpose are complementary genomic target sequence and/or zone with it. For example, if purpose is to screen in the biological sample (for example blood, respiratory tract material, liver, lung) whether have SARSV, preferably with oligomer as probe and/or primer and with itself and the genomic conservative area hybridization of SARSV. The combinative SARSV of described oligomer narrates in more genomic conservative zones in this article, for example 5 ' UTR and 3 ' UTR.

In basic nucleic acid hybridization test, strand analyte nucleic acid (DNA or RNA) and nucleic acid probe hybridization, and detect the gained duplex. The length of SARSV polynucleotides (natural or extend) probe can be by the virus sequence of hybridization check uniqueness. Although 6-8 nucleotides is the length that can work, the sequence that contains 10-12 nucleotides is preferred, contains to have an appointment 13,14,15,16,17,18,19,20 or 21 or more polynucleotides are seemingly best. Preferred these sequences will be from lacking heterogeneous zone. Available conventional method prepares these probes, comprising automatic oligonucleotides synthetic method. Useful probe for example can be those derived from the SARSV genome probe in very conservative zone not. Be not difficult determine in SARS genome very not conservative zone by sequence contrast and other technology of knowing of providing here. Also satisfactory with the sequence of any differentiated part complementation of SARSV genome. For as probe, complete complementary preferably, but this is optional when fragment length increases.

There is a situation (for example screen contaminated blood or diagnose infected individuality) for what detect the SARSV polynucleotides with this probe, can processes to extract wherein contained nucleic acid to biological sample to be analyzed (including but not limited to blood, serum, lung, liver, mucous membrane, kidney, saliva or phlegm) if need. Can carry out gel electrophoresis or other technology of separating according to molecular size range to gained sample nucleic acid; Perhaps, can carry out Dot blot and need not to separate by size nucleic acid. For the target-seeking sequence with probe forms the heteroduplex body, the target region of nucleic acid analyte must be single stranded form. To not need to carry out sex change when existing with single stranded form when this sequence is natural. Yet, when sequence exists with double chain form, need this sequence sex change. Sex change can be carried out with various techniques known in the art. After the sex change, the target sequence of supposing in nucleic acid analyte and the probe target sequence in promoting probe and the analyte formed under the condition of stablizing crossbred cultivate, and the detection gained contains the duplex of probe.

The detection of gained duplex (if existence) is finished with the probe of mark usually; Perhaps, probe can not be labeled yet, but can directly or indirectly detect by being combined with the ligand specificity who is labeled. The method of suitable label and label probe and part is known in the art, comprising for example radioactive label (available known method (for example nick translation or kinases) mixes), biotin, fluorophor, chemiluminescent groups (for example dioxetane, the especially dioxetane of triggering), enzyme, antibody etc.

Being used for the probe area of bound analyte can be made into and SARSV genome complete complementary. Therefore, usually need high stringent condition to prevent false positive. Yet, only have when probe and just need to use high stringent condition when lacking heterogeneous viral genome regional complementarity. The stringency of hybridization is to be determined by the many factors in crossover process and the washing process, comprising temperature, ionic strength, time length and formamide concentration. These factors are listed in lists Maniatis T. (1982) in.

Also can use the variation of this basic methods, these variations are known in the art, are conducive to separate the variation of duplex to be detected and/or from the variation of the part amplifying signal of mark from allogenic material comprising those. Can consult for understanding these changes, for example: Matthews and Kricka (1988), Analytical Biochemistry 169:1; Landegren etc., (1988), Science 242:229; And Mittlin (1989), Clinical Chem. 35:1819. These documents and the following mode determination of openly having described are included into this paper as a reference. The probe that is adapted in these assay methods detecting SARSV comprises and the hybridization of target SARSV polynucleotide sequence forms sequence with analyte chain duplex, and wherein this duplex enough is stablized with to be detected in described detection system.

A suitable variation is described in, for example, U.S. Patent No. 4,868, the open No.225 of 105 (submissions on September 9th, 1989) and EPO is in 807 (on June 16th, 1987 is open). These have openly described a kind of solution phase nucleic acid hybridization detection method, wherein, and nucleic acid analyte and label probe group and the hybridization of capture probe group. Probe-analyte complex is by the coupling with capture probe (complementary with the capture probe group) hybridization of solid support. Nucleic acid analyte can be removed from solution as the solid phase complex like this. Analyte is that solid phase complex form is conducive in this determination method separating step subsequently. The label probe group is with complementary by the probe of the mark of combination with solid phase/analyte complex hybridization.

PCR (PCR) is the technology of contained required nucleotide sequence (target) or its mixture in a kind of amplification of nucleic acid. In PCR, use the complementary strand hybridization of a pair of excessive primer and target nucleic acid. Primer will extend under the effect of polymerase as template separately with target nucleic acid. Extension products self becomes again target sequence, then dissociates from the initial target chain. Then new primer is hybridized and at the effect downward-extension of polymerase, and this circulation is repeated to increase with how much level ground the quantity of target sequence molecules. PCR is disclosed in United States Patent(USP) Nos. 4,683, and 195 and 4,683,202, they are included into this paper as a reference.

Ligase chain reaction (LCR) is the another kind of method of nucleic acid amplification. In LCR, use the probe pair contain two kinds of one-level (first and second) probes and two kinds of secondarys (third and fourth) probe, these probes all use with the mole that surpasses target. The first section hybridization of the first probe and target chain, the second section hybridization of the second probe and target chain, the first and second sections adjoin, thus the one-level probe mutually adjoin with the relation of 5 ' phosphoric acid-3 hydroxyl, thereby ligase can merge these two probe covalency or be connected to form fusion product. In addition, the 3rd (secondary) probe can be hybridized with the part of the first probe, and the 4th (secondary) probe can similarly adjoin the part hybridization of mode and the second probe. Certainly, double-stranded if target is initially, then the secondary probe also will be hybridized with the complementary strand of target in the first situation. In case the one-level probe chain that connects separates with the target chain, it will with the third and fourth Probe Hybridization, make them be connected to form complementary secondary and connect product. Importantly to recognize, connect product and target or its complement function equivalence. Repeatedly hybridize and be connected circulation and can realize the amplification of target sequence by many wheels. This technology more is described in detail the EP-A-0439182 (on July 31st, 1991 disclosed) of EP-A-320 308 in K.Backman (on June 16th, 1989 is open) and K.Backman etc., and they are included into this paper as a reference.

For amplification mRNA, be that the mRNA reverse transcription is become cDNA within the scope of the present invention, then by PCR (RT-PCR) amplification; Or in two steps, all adopt single enzyme, and such as U.S. Patent No. 5,322,770 is described, and this patent is originally included this paper in as a reference; Or the mRNA reverse transcription become cDNA, then carry out asymmetric nick-joining enzyme chain reaction (RT-AGLCR), such as [PCR Methods and Applications 4:80-84 (1994)] as described in R.L.Marshall etc., also be included into this paper as a reference.

TMA is described in detail in, for example, U.S. Patent No. 5,399,491, its content is included in this paper as a reference in full. In the embodiment of a conventional determining method, the nucleic acid samples that suspection is contained the separation of SARSV target sequence mixes with the buffering concentrate that contains buffer solution, salt, lintel, nucleotides triphosphoric acid, primer, dithiothreitol (DTT) and spermidine. Reactant chosen wantonly at about 100 ℃ lower cultivate about 2 minutes so that all secondary structure sex change. After being cooled to room temperature, add reverse transcriptase, RNA polymerase and RNA enzyme H, and mixture was cultivated 2-4 hour at 37 ℃. Then can be by following process assaying reaction: make the product sex change, add probe solution, cultivated 20 minutes at 60 ℃, add can selective hydrolysis the solution of hybridization probe not, remaining chemiluminescence was cultivated 6 minutes and measured to reactant at 60 ℃ in photometer.

Usually, TMA may further comprise the steps: (a) from suspecting the interested biological sample isolating nucleic acid that is infected by SARSV, comprise RNA; (b) following material is mixed the formation reactant mixture: the nucleic acid that (i) separates, (ii) the first and second Oligonucleolide primers, the first primer has and compound RNA target sequence is (if present with it, (+) chain for example) the abundant complementary complex sequence of 3 ' end portion, the second primer has and the fully complementary complex sequence of 3 ' end portion of the target sequence of compound complement (for example (-) chain) with it, wherein said the first oligonucleotides also contains the 5 ' sequence to described complex sequence, wherein comprise promoter, (iii) reverse transcriptase or RNA and DNA dependent dna-polymerases, (iv) enzymatic activity of the RNA chain of degradation selectivity RNA-DNA complex (such as RNA enzyme H) and (v) can identify the RNA polymerase of this promoter.

The component of reactant mixture can progressively be mixed or disposable mixing. Reactant mixture is cultivated the sufficiently long time so that the multicopy of target sequence to be provided under the condition that forms oligonucleotides/target sequence (condition (comprising ribonucleotide triphosphate and DNA triphosphoric acid) that comprises initiation DNA and nucleic acid). Reaction should occur under the condition that is fit to keep the reactive component stability such as enzyme component, and does not need to change in the amplified reaction process or the control reaction condition. Therefore, reaction can occur under basic isothermal and ionic strength and the substantially invariable condition of pH. This reaction does not need denaturing step to separate the RNA-DNA complex of for the first time DNA extension generation usually.

Suitable archaeal dna polymerase comprises reverse transcriptase, such as AMV (AMV) reverse transcriptase (available from for example Seikaaku America, Inc.) and MMLV (MMLV) reverse transcriptase (available from for example Bethesda Research Laboratories).

Be fit to mix the promoter of primer or promoter sequence by the nucleotide sequence of RNA polymerase specific recognition (can be natural generation, that make by synthetic method or the product of restrictive diges-tion), this enzyme identification and in conjunction with this sequence and start transcription, thus make the rna transcription thing. This sequence can be chosen wantonly and contain the nucleotide base that extends beyond the actual recognition site of RNA polymerase, can increase like this stability of degradation treatment or sensitiveness or raising are transcribed efficient. The example of useful promoter comprises that those are by the promoter of some bacteriophage polymerase identification, such as bacteriophage T3, T7 or SP6 promoter or escherichia coli promoter. These RNA polymerases can obtain by commercial source, such as New England Biolabs and Epicentre.

Some reverse transcriptases that are suitable for method described here have RNA enzyme H activity, such as the AMV reverse transcriptase. Yet, should add exogenous RNA enzyme H, such as e. coli rna enzyme H, when namely box lunch uses the AMV reverse transcriptase. RNA enzyme H can available from, for example, Bethesda Research Laboratories.

The rna transcription thing that makes with these methods can be used as template to make other copy of target sequence by above-mentioned mechanism. This system be self-catalysis and increase by self-catalysis, do not need to repeat to modify or change the reaction conditions such as temperature, pH, ionic strength.

Detection can in all sorts of ways and finish, comprising direct Sequencing, with the hybridization of sequence-specific oligomer, gel electrophoresis and mass spectrum. These methods can adopt heterogeneous or the homogeneity pattern, isotope or heterotope mark, or just do not have mark at all.

Include, but are not limited to the primer that is used for the inventive method and/or the suitable label that probe is combined: 5-FAM (is also referred to as CF; Be also referred to as spiral shell (isobenzofuran-1 (3H), 9 '-(9H) xanthene)-5-carboxylic acid, 3 ', 6 '-dihydroxy-3-oxo-6-Fluoresceincarboxylic acid); 5-chlordene-fluorescein ([4,7,2 ', 4 ', 5 ', 7 '-chlordene-(3 ', 6 '-dipivalyl fluorescein base)-6-carboxylic acid]); 6-chlordene-fluorescein ([4,7,2 ', 4 ', 5 ', 7 '-chlordene-(3 ', 6 '-dipivalyl fluorescein base)-5-carboxylic acid]); 5-tetrachloro-fluorescein ([4,7,2 ', 7 '-tetrachloro-(3 ', 6 '-dipivalyl fluorescein base)-5-carboxylic acid]); 6-tetrachloro-fluorescein ([4,7,2 ', 7 '-tetrachloro-(3 ', 6 '-dipivalyl fluorescein base)-6-carboxylic acid]); Tetramethylrhodamin (TAMRA) comprises (i) 5-TAMRA (5-carboxyl tetramethylrhodamin; Xanthylium, 9-(2,4-dicarboxyl phenyl)-3,6-two (dimethylaminos) and (ii) 6-TAMRA (6-carboxyl tetramethylrhodamin; Xanthylium, 9-(2,5-dicarboxyl phenyl)-3,6-two (dimethylamino); EDANS (5-((2-aminoethyl) amino) naphthalene-1-sulfonic acid); 1,5-IAEDANS (5-((((2-iodo acetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid); DABCYL (4-((4-(dimethylamino) phenyl) azo) benzoic acid); Cy5 (iodo two carbocyanines-5); Cy3 (iodo two carbocyanines-3); And BODIPY^TMFL (4,4-, two fluoro-5,7-dimethyl-4-boron-3a, 4a-diaza-s-indacene-3-propionic acid). Preferably use simultaneously FAM (for example at 5 ' mark) and TAMRA (for example at 3 ' mark) label probe.

Nucleic acid of the present invention can the solution form uses or is combined with solid matrix or holder, for example with the pattern of DNA array.

Obvious, the design of assay method described here can have multiple variation and various modes, and this is known in the art. Top narration is only as a kind of guidance, and the technical staff who is proficient in this field can use technology well known in the art easily described method to be improved.

A kind of 302nt amplicon of SARS virus is called as " BNI-1 " (SEQ ID NO:9927). At the Bernhard of Hamburg, Germany Nocht Institute it is checked order. In April, 2003, the BNI-1 sequence comes forth and WHO website (http://www.who.int/csr/sars/primes/en/) and Dorsten etc., " identify a kind of novel coronavirus in the severe acute respiratory syndrome patient " (Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome), NewEngland Journal of Medicine is published in http://www.nejm.org online. These two reference treatment are included in this paper as a reference in full. Embodiments more of the present invention do not comprise the nucleic acid that contains SEQ ID NO:9927. Some of the other embodiments of the present invention does not comprise the nucleic acid that contains SEQ ID NO:9927. Embodiments more of the present invention do not comprise and contain SEQ ID NO^s: arbitrary nucleic acid among the 9928-9959. Some of the other embodiments of the present invention does not comprise and contains SEQ ID NO^s: arbitrary nucleic acid among the 9928-9959. Embodiments more of the present invention do not comprise these exclusive segments.

Immunoassays

The present invention has used various immunoassaies to identify the individuality of contacted SARSV, and/or contains the biological sample of SARSV or SARSV antibody.

The immunoassays pattern

In fact, SARSV antigen can be used for the detecting pattern that any employing known antigens detects antibody. All these detect a common trait, make under antigen and the condition of suspecting any this antibody-like that the biological sample that contains SARSV antibody exists in permission antigen is combined component to contact. This condition is physiological temp, pH and ionic strength normally, and uses excessive antigen. Antigen cultivated with sample then detect the immunocomplex that is consisted of by antigen. Perhaps, can come the SARSV antigen that exists in the detection of biological sample with anti--SARSV antibody. Also antigen/antibody can be detected and combine; For example, such as United States Patent (USP) 6,630, the HCV described in 298 detects.

The immunoassays pattern be designed with many variations, and various modes is arranged, this is known in the art. For example, scheme can be used solid support or immunoassays effect. Many determination methods have been used antibody or the polypeptide of mark; Mark can be, for example, and enzyme labeling, fluorescence labeling, chemiluminescent labeling, radioactive label or dye molecule. Also the determination method of known amplification immune complex signal is for example used the determination method of biotin and avidin and the immunoassays of enzyme labeling and mediation, measures such as ELISA.

Immunoassays may be, but not limited to,, heterogeneous or homogeneity pattern, and can be type or type of competition. In heterogeneous pattern, polypeptide is combined so that after cultivating sample is separated with polypeptide with solid matrix or holder. The example of operable solid support comprises that NC Nitroncellulose (for example, be fixed on the film or the microtitration well format), polyvinyl chloride (for example, in lamella or microtiter well), polystyrene latex (for example, in microballon or the microtiter plate), poly-partially (two) PVF (polyvinylidine fluoride), diazotising paper, nylon membrane, microchip, high density or low-density biochip, recombinant immune measure (RIBA), Micro Fluid device, little magnetic bead, activated beads and a-protein pearl. For example, the polystyrene bead (Precision Plastic Ball) of Dynatech Immunlon or Immunlon 2 microtiter plates or 0.25 inch can heterogeneous pattern using. The solid support that will contain antigenic polypeptide separates the rear washing that usually needs with test sample, and then detects the antibody of combination. Mode standard and competitive pattern all are known in the art.

In the homogeneity pattern, test sample and antigen mixture are cultivated in solution. For example, can under the condition that will precipitate formed any antigen-antibody complexes, cultivate. The mode standard of these mensuration and competitive pattern are known in the art.

In dimension model, the amount of SARSV antibody in antibody-antigenic complex will be monitored directly. This can-SARSV antibody epitope anti-by identification of determining mark anti--allogene (for example, Anti-Human) antibody whether owing to form complex in conjunction with finishing. In competitive pattern, can reason out by the competitive effect of the antibody (or other competition part) of the mark of known quantity in the monitoring complex amount of SARSV antibody in the sample.

Can be by in numerous known technologies any, according to the pattern that adopts, detect the formed complex that contains anti--SARSV antibody (in the competitive assay for detecting the amount of competition antibody). For example, the conjugate of the compound anti-allogene Ig of available and mark (for example enzyme labeling) detects unlabelled SARSV antibody in the complex.

In immunoprecipitation or CA pattern, SARSV antigen and antibody response form the network that precipitates from solution or suspension, form the visible beds of precipitation or precipitation membrane. If nonreactive in the test sample-SARSV antibody then can not form visual precipitation.

Particle agglutination (PA) is measured has three kinds of particular types at least. These determination methods are used to detect the antibody that is coated on the various antigens on the holder. One type of this determination method is that hemagglutination is measured, use by with antigen (or antibody) passive adsorption to red blood cell (RBC) and the RBC of sensitization. If there is the antibody of other specific antigen during health forms, then can make the RBC cohesion that is coated with purifying antigen.

For eliminating the nonspecific reaction that may exist in the hemagglutination test, available two kinds of artificial carriers replace RBC in PA. Referring to be latex particle. Yet also can use gelatin particle. Use the mensuration any in these particles can be based on the passive agglutination of the particle that scribbles purifying antigen.

SARSV antigen is packaged into the form for the kit of these immunoassays usually. These kits usually contain the required labelled antibody of the natural SARSV antigen that places autonomous container, control antibodies preparation (positive and/or negative), mode determination and also contain signal propellant (such as zymolyte) when mark directly do not produce signal. Natural SARSV antigen can be combined with solid matrix or be separated with the reagent of binding matrix. Usually also comprise the explanation (such as paper spare, tape, CD-ROM etc.) of measuring in the kit.

Use the immunoassays of natural SARSV antigen also to be used to screen there is not latent infection SARASV in blood with preparation blood supply. The method for preparing blood supply may further comprise the steps. Blood donor's health is formed, and preferred blood or blood constitutent contact with natural SARSV antigen, so that between SARSV antibody (if existence) and the SARSV antigen immune response occurs. Whether detection reaction forms anti-SARSV antibody-SARSV antigenic complex. The donor blood that natural SARSV antigen-antibody do not occur forms blood supply.

The antibody preparation

As mentioned above, mensuration can be used the various antibody that are incorporated into solid support, detectable antigens or formed antigen/antibody complex when existing SARSV to infect in the sample. These antibody can be polyclone or monoclonal antibody goods, monospecific antiserum, and people's antibody perhaps can be hybridization or chimeric antibody, such as humanized antibody, variation antibody, F (ab ')₂Fragment, F (ab) fragment, the Fv fragment, single domain antibody, dimerization or trimerization antibody fragment construction, microbody, or in conjunction with its functional fragment of antigen to be checked.

Antibody is to make with the technology known by the technical staff of being proficient in this field, for example, and United States Patent(USP) Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; With 4,372,745. For example, polyclonal antibody is by producing with the suitable animal of interested antigen immune (such as mouse, rat, rabbit, sheep or goat). For strengthening immunogenicity, can antigen be attached on the carrier then in immunity first. This carrier is that one of ordinary skill in the art is known. Immunity is carried out usually like this: antigen is mixed or emulsification with salt solution (better with adjuvant such as Freund's complete adjuvant), and then parenteral (normally subcutaneous or intramuscular) is injected this mixture or emulsion. The protein of using afterwards salt solution (preferably using incomplete Freunds adjuvant) to join in 2-6 week is injected one or many with booster immunization. Can carry out external immunity with methods known in the art in addition and produce antibody. Then the animal from immunity obtains polyclonal antiserum.

As indicated above, method [(1975) Nature of the common available Kohler of monoclonal antibody and Milstein256: 495-497] or its manufacturing of improving one's methods.

As mentioned above, the antibody fragment that can identify SARS antigen is also included within the scope of the invention. Many antibody fragments that contain the antigen binding site that can show complete antibody molecular immune binding characteristic are known in the art. For example, can be by producing F (ab ') with Pepsin digestion for example except the constant region of not responsible antigen combination in the antibody molecule₂Fragment is come the manufacturing function antibody fragment. These fragments will contain two antigen binding sites, but lack the part constant region of every heavy chain. Similarly, if necessary, can make the Fab fragment that contains single antigen binding site, for example, by using papain digestion polyclone or monoclonal antibody. Also but the Application standard technology is made the functional fragment of the variable region that only comprises heavy chain and light chain, these technology such as recombination method or preferential proteolysis cutting immunoglobulin molecules. These fragments are called as F_v Referring to, for example, Inbar etc., (1972) Proc.Nat.Acad. Sci USA69: 2659-2662; Hochman etc., (1976) Biochem15: 2706-2710; And Ehrlich etc., (1980) Biochem19：4091-4096。

ScFv (" sFv " or scFv ") polypeptide is a kind of covalently bound V_H-V _LHeterodimer, it is the V that connects by peptide coding joint by comprising_H-and V_LThe fusion of-encoding gene is expressed. Huston etc., (1988) Proc.Nat.Acad.Sci.USA85: 5879-5883. Describe many resolutions and set up the method for chemical constitution (joint), these structures are used for natural cohesion but the light and heavy polypeptide chain in the antibody V zone that can separate by chemical means changes the sFv molecule into, and the sFv molecule will be folded into and the substantially similar three-dimensional structure of structure of antigen binding site. Referring to, for example, United States Patent(USP) Nos. 5,091,513; 5,132,405; With 4,946,778. Can be used on the method for having described this area and make the sFv molecule. Referring to, for example, Huston etc., (1988) Proc.Nat. Acad.Sci USA85: 5879-5338; United States Patent(USP) Nos. 5,091,513; 5,132,405 and 4,946,778. Design standard comprises the substantial distance of distance between the N-end of terminal and another chain of the C-that determines a chain, and wherein said joint is formed by the little hydrophilic amino acid residue that can not be curling or forms secondary structure usually. This method was described in this area. Referring to, for example, United States Patent(USP) Nos. 5,091,513; 5,132,405 and 4,946,778. Suitable joint generally includes the polypeptide chain that glycine and serine are arranged alternately, and can comprise that the glutamic acid of insertion and lysine residue are to strengthen dissolubility.

" small antibody " or " microbody " also can be used for the present invention. Microbody is the sFv polypeptide chain, comprises the oligomerization zone of separating by hinge region and sFv at their C-end. Pack etc., (1992) Biochem31: 1579-1584. The oligomerization zone comprises that for example leucine zipper can make it more stable by other disulfide bond from uniting alpha-helix. The oligomerization zone is designed to be complementary with the cross-film directionality is folding, and this process is considered to be conducive to be folded in the polypeptide body functional in conjunction with albumen. Usually, microbody is with recombination method preparation well known in the art. Referring to, for example, Pack etc., (1992) Biochem31: 1579-1584; Cumber etc., (1992) J.Immunology149B：120-126。

Make SARS antigen

Normally make by recombination method for SARSV antigen of the present invention. Therefore, the polynucleotides available standards Protocols in Molecular Biology that is used for encoding SARS V antigen of the present invention is made. For example, the polynucleotide sequence of the above-mentioned molecule of encoding can obtain with recombination method, such as cell screening cDNA and the genomic library from expressing gene, or derives this gene from the known carrier that contains this gene. In addition, can be used on the technology that this area was described, the technology that is used for HCV described in the U.S. Patent No. 5,350,671 of Houghton etc. is directly separated required gene from the viral nucleic acid molecule. Except the clone, the gene of the antigen interested of encoding also can be made by synthetic. The suitable codon of available particular sequence (the selected host's of preferred expression best codon) designs molecule. Complete sequence is normally assembled from the overlapping oligonucleotides by the standard method preparation, and is assembled into complete coded sequence. Referring to, for example, Edge, Nature (1981) 292:756; Nambair etc., Science (1984) 223:1299; Jay etc., J.Biol.Chem. (1984) 259:6311.

Therefore, can obtain specific nucleotide sequence from carrier, contain required sequence in this carrier or with all or part of synthetic sequence of various oligonucleotides synthetic technologys known in the art (comparatively suitable have direct mutagenesis and PCR (PCR) technology). Referring to, for example, Sambrook, the same. Specifically, a kind of method of nucleotide sequence of the required sequence that obtains to encode is the complement group annealing that makes the overlapping oligonucleotides of synthetic property of making in conventional automation polynucleotides synthesizer, then connects with suitable dna ligase and the nucleotide sequence by the pcr amplification connection. Referring to, for example, Jayaraman etc., (1991) Proc.Natl.Acad.Sci.USA 88:4084-4088. In addition, the present invention also can adopt the directed synthetic (Jones etc. of oligonucleotides, (1986) Nature 54:75-82), oligonucleotides directed mutagenesis (the Riechmann etc. in existing oligonucleotides zone, (1988) Nature 332:323-327, with Verhoeyen etc., (1988) Science 239:1534-1536), and fill gapped oligonucleotides (Queen etc. with the T4DNA polymerase by enzyme, (1989) Proc.Natl.Acad.Sci.USA 86:10029-10033), change or the antigen binding capacity of enhancing and/or the immunogenic molecule of reduction to provide to have.

In case coded sequence is produced or separates, this sequence clone can be entered in any suitable carrier or replicon. Be proficient in the known many cloning vectors of technical staff in this field, selecting suitable cloning vector is a kind of selection problem. Suitable carrier includes, but not limited to plasmid, bacteriophage, transposons, clay, chromosome (comprising the artificial chromosome, such as BAC or YAC) or when the virus that can copy when suitable control element is combined.

Then coded sequence is placed under the suitable control element control system that this depends on to express. Therefore, coded sequence is under promoter, ribosome bind site (the being used for bacterial expression) control, and optional it is under the operon control, thereby interested dna sequence dna is transcribed into RNA by suitable transformant. Coded sequence can contain or not contain signal peptide or targeting sequencing, and this sequence is processed after subsequently can the translation by the host and is removed. Referring to, for example, United States Patent(USP) Nos. 4,431,739; 4,425,437; 4,338,397.

Except control sequence, can also add regulating and controlling sequence is be proficient in this field known to the skilled with the expression regulation sequence according to the growth regulating sequence of host cell, and its example comprises the sequence that those open or close gene expression according to chemistry or physical stimulation (comprise existing and regulate compound). The controlling element that also can have other type in the carrier. For example, can use enhancer element to improve the expression of construction here. Its example comprises SV40 early gene enhancer (Dijkema etc., (1985) EMBO is J.4:761), from the enhancers/promoters (Gorman etc. of Rous sarcoma virus LTR (LTR), (1982) Proc.Natl.Acad.Sci.USA 79:6777) and from the element (Boshart etc. of people CMV, (1985) Cell 41:521), such as element (U.S. Patent No. 5 contained in the CMV intron A sequence, 688,688). Also can be included in one or more selectable markers, one or more restriction site, high copy number potential and the strong promoter of the spontaneous replication initiation sequence that copies in the suitable host cell in the expression cassette.

Having made up a kind of expression vector makes the specific coding sequence be arranged in the carrier that contains suitable regulating and controlling sequence, relative control sequence, the position of coded sequence and direction should be able to make this coded sequence be transcribed (that is, RNA polymerase is transcribed this coded sequence in conjunction with the control sequence of dna molecular) under " control " of control sequence. May need the sequence of coding molecules of interest is modified to reach final purpose. For example, must modify with can be with suitable orientation in conjunction with control sequence sequence in some situation; Namely in order to keep reading frame. Can be before insertion vector with control sequence be connected regulating and controlling sequence and be connected with coded sequence. Perhaps, the coded sequence Direct Cloning can be entered to contain the expression vector of control sequence and suitable restriction site.

As mentioned above, may need to make mutant or the analog of antigen interested. Its method is described in, for example, and Dasmahapatra etc., U.S. Patent No. 5,843,752 and Zhang etc., U.S. Patent No. 5,990,276. Partial sequence or insetion sequence that can be by deletion coding polypeptide of interest and/or replace one or more nucleotides in the sequence and make SARSV mutant or analog for described mensuration. The technology of modified nucleotide sequence such as direct mutagenesis etc., is be proficient in this field known by the technical staff. Referring to, for example, Sambrook etc., the same; Kunkel, T.A. (1985) Proc.Natl.Acad.Sci.USA 82:448; Geisselsoder etc., (1987) BioTechniques 5:786; Zoller and Smith (1983) Methods Enzymol.100:468; Dalbie-McFarland etc., (1982) Proc.Natl.Acad.Sci USA 79:6409.

Molecule is expressed in can be in many systems, comprises insect, mammal, bacterium, virus and yeast expression system, and these all are well known in the art.

For example, insect cell expression system, such as rhabdovirus system, be proficient in this field known to the skilled, and be described in, for example, Summers and Smith, " De Kesaxin agricultural experiment present situation communique " (Texas Agricultural Experiment Station Bulletin) No.1555 (1987). Be used for form that the materials and methods of baculoviral/insect cell expression system can kit available from Invitrogen, San Diego Calif. (" MaxBac " kit). Similarly, bacterium and mammalian cell expression system be well known in the art and be described in, for example, Sambrook etc., the same. Yeast expression system also is known in the art, and is described in, for example, and " yeast genetic engineering " (Yeast Genetic Engineering) (volume such as Barr, 1989) Butterworths, London.

Many suitable host cells for said system also are known. For example, mammal cell line is known in the art and comprises immortal cell line available from American Type Culture Collection (ATCC), for example, but be not limited to, Chinese hamster ovary (CHO) cell, HeLa cell, young hamster kidney (BHK) cell, MK cells (COS), human embryonic kidney cell, human liver cell cancer cell are (for example, Hep G2), Madin-Darby ox kidney (" MDBK ") cell, and other cell. Similarly, the bacterial hosts such as Escherichia coli, bacillus subtilis and streptococcus also can be used for expression construction of the present invention. Be used for yeast host of the present invention and comprise saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), maltose Candida (Candida maltosa), Hansenula polymorpha (Hansenuala polymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Pichia guillerimondii, pichia pastoris phaff (Pichia pastoris), fission yeast (Schizosaccharomyces pombe) and Yarrowia lipolytica (Yarrowia lipolytica). The insect cell that is used for rhabdovirus expression vector comprises Aedes aegypti (Aedes aegypti), autographa california (Autographa californica), silkworm (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), fall army worm (Sodoptera frugiperda) and broccoli looper (Trichoplusia ni).

Use range gene conveying technology well known in the art the nucleic acid molecules stable integration that contains nucleotide sequence interested can be entered the host cell gene group or in suitable host cell, maintain stable episome element. Referring to, for example, U.S. Patent No. 5,399,346.

According to selected expression system and host, can make molecule by the host cell that is transformed by above-mentioned expression vector is grown under the condition of marking protein. The protein of then separate expressing from host cell also is purified. If expression system is secreted into protein in the growth medium, can be directly from the culture medium purified product. If non-secretory, then can be from the cell lysate separated product. Selection to suitable growth conditions and recovery method is be proficient in this field known to the skilled.

Embodiment

Be effective expression SARSV antigen in saccharomyces cerevisiae (Saccharomyces cerevisiae) and pichia pastoris phaff (Pichia pastoris), insect cell and mammalian cell, the domain that following table is listed is cloned into expression vector. The nt sequence number is from the SARSV sequence of SEQ ID NO:1.

-RNA polymerase 1a:SARS nt 250-13398

-RNA polymerase 1b:SARS nt 13399-21470

-ORFns. coating (with ns2, hemagglutinin-esterase envelope glycoprotein and spike glycoprotein homology): SARS nt 21477-25244

-film: SARS nt 27849-28103

-nucleocapsid: SARS nt 28105-29373

Combination with PCR and synthetic oligomer produces the said structure territory, connects into following expression vector by restriction site:

The two ends carrier promoter expression host of restriction site

HindIII/SaiI pBS24.1 ADH2/GAPDH AD3/ saccharomyces cerevisiae

EcoRI/Sal|| pBS24.1 ADH2/GAPDH/SOD merge the AD3/ saccharomyces cerevisiae

XbaI/SalI pAO815 AOXI GS115/ pichia pastoris phaff

The EcoRI/BamHI pCMVkm2 CMVp/ enhancer/of short duration transfection of intron A HVK-293/

EcoRI/XmaI pCMVIII CMVp/ enhancer/intron A CHO stable cell lines

The clone that Chiron adopts,

NheI/SalI pBluBac4.5 polyhedrin comprises: Sf9, Sf21, Tn5

IV. infect with RNAi treatment SARS

RNA disturbs or " RNAi " is the initial term of being created by Fire and its colleague, the phenomenon (Fire etc., Nature 391,806-811 (1998)) of gene expression capable of blocking when being used for describing with double-stranded RNA (dsRNA) introducing worm. The RNAi most probable relates to the degraded of mRNA, and causes sequence-specific PTGS in many organisms. RNAi is a kind of rear processing of transcribing that double-stranded RNA causes of introducing, and causes gene silencing in the sequence-specific mode. It is reported that RNAi can produce naturally in the multiple species such as nematode, trypanosome, plant and fungi. It is protected possibly, and organism exempts from virus infections, the regulating rotary stand is active and get rid of unusual transcription product.

DsRNA can realize that the first evidence of effective gene silence is from the research (Fire etc. to Caenorhabditis elegans (Caenorhabditis elegans) by RNAi, (1998) Nature, 391:806-811 and U.S. Patent No. 6,506,559). The RNAi that studies confirm that in Drosophila melanogaster (Drosophila melanogaster) is a kind of two step mechanism (Elbashir etc., (2001) Genes Dev., 15 (2): 188-200) subsequently. At first, long dsRNA is called the fragment that the enzyme of cutting enzyme cuts into 21-23 nucleotides (nt) by a kind of, is called siRNA (siRNA). Then, siRNA and ribonucleic acid combined enzyme agent (being called RISC, i.e. the RNA silencing complex of inducing) combination makes the mRNA of this complex target complementation. Then RISC cuts the complementary siRNA of said target mrna opposition, makes this mRNA responsive to other RNA degradation pathway.

RNAi is a kind of phenomenon, and at this moment, the dsRNA corresponding with target DNA or RNA sequence can suppress or cryptiogene is expressed. Even if but the intracellular gene specific of dsRNA mediate mammalian disturbs (Wianny and Zernicka-Goetz (2000) Nature Cell Biol.2:70-75 in some cases; Svoboda etc.; (2000) Development 17:4147-4156); but in mammalian somatic cell, use RNAi normally limited; this is because dsRNA causes the protein kinase (PKR) that ds depends on RNA; and then deactivation translation factor eIF2a causes the synthetic comprehensive inhibition of protein, and often can cause Apoptosis (Gil and Esteban (2000) Apoptosis 5:107-114).

Adopt this year the siRNA corresponding to about 21 or 22 base-pairs of the length of target RNA or dna sequence dna to carry out the gene specific inhibition, demonstration can be interrupted the expression (Elbashir of these target sequences in mammalian cell, S.M. etc., Nature 411:494-498 (2001)). Yet whether genomic all RNA of not clear mammalian cell or DNA sequence be all responsive to siRNA. And fail to determine whether that every kind of mammalian cell types all has with siRNA causes gene specific to suppress necessary mechanism. In addition, restriction uses siRNA to have following two reasons at least: the of short duration characteristic of observing this depression effect in using the cell of siRNA; And in some cases must be before using chemical synthesis siRNA (Tuschl T., Nature Biotechnol., 20:446-448 (2002)). Simultaneously, these short synthetic RNA are also unstable so that these siRNA are become problem as long-time medicine.

For overcoming these restrictions, the invention provides a kind of nuclease degradation that can resist and improve stable it disturbs the modification that suppresses Virus reproductivity by RNA the siRNA that still keeps simultaneously. This modification to the siRNA ribonucleotide is to add a chemical group by chemical synthesis or in-vitro transcription, and perhaps one of available these methods prepare the RNA of long modification and are cut to siRNA with cutting enzyme.

Although other gene specific inhibition method has been used the nucleic acid of chemical modification, such as antisense technology and ribozyme technology, this modification has destroyed and has made these technology necessary key enzymatic activity that plays a role. With regard to antisense technology, it is active that the modification of ribonucleotide has been destroyed RNA enzyme H, and such modification will be abolished the catalytic activity of ribozyme.

The invention provides a kind of double-stranded RNA that can resist nuclease degradation (dsRNA) molecule through modifying, its length is about 10-30 nucleotides, can be in mammalian cell inactivation of viruses. It is a kind of by giving to come through the siRNA (siRNA) of modifying the method for inactivation of viruses that the present invention also provides, described siRNA process is modified so that they have ribozyme or RNA enzyme resistance, and keeps the BA that can suppress by the RNA sequence of target-seeking virus virus replication.

The invention still further relates to the method for the modification siRNA that makes target viral RNA sequence, the method comprises preparing at least one crosses over the double-stranded RNA (dsRNA) of the modification of the ribonucleotide that contains at least one modification in the virus genomic chain; And cut dsRNA fragment that enzyme cutting modifies with recombined human and obtain the siRNA that modifies more than one.

The invention provides the dsRNA molecule of the modification that contains 10-30 the nucleotides of having an appointment, described molecule can mediate target RNA and disturb in liver cell or SRAS infection cell.

RNA disturbs or RNAi represents the expression (protein is synthetic) of sequence-specific or gene specific different plasmagene in this article, and can not cause the synthetic comprehensively inhibition of the cell protein that contains this siRNA. The present invention is not subjected to the concrete theoretical restriction of RNAi mechanism of action. For example, RNAi may participate in the degraded of mRNA (mRNA) in the silencing complex (RISC) that RNA induces, stops the mRNA translation of transcribing, and what perhaps it may participate in genomic DNA methylates, changes transcribing of gene. The gene expression shortage that RNAi causes can be temporary transient, the lasting short period or can be period stable, permanent, that continue endless.

Term RNA has art-recognized implication. In addition, RNA also is used in this article representing double-stranded RNA (dsRNA) or single stranded RNA (ssRNA) or contains the dsRNA of strand jag. DsRNA represents short interfering rna (siRNA), Microrna (miRNA) and bobby pin RNA (shRNA) in the present invention, in addition, RNA also is used for representing mRNA (mRNA), transfer RNA (tRNA) or rRNA (rRNA).

The present invention relates to through the siRNA (siRNA) of chemical modification with nuclease-resistant stability to degradation that enhancing is provided, but these siRNA still can be in conjunction with the target RNA that may exist in the cell. When target RNA was virus-specific RNA, the siRNA of modification can be in conjunction with virus-specific RNA and inactivation of viruses. The siRNA of modification of the present invention contains the ribonucleotide of modification, and wherein said siRNA can resist the enzyme degraded, such as the degraded of RNA enzyme, and still keeps the ability that suppresses virus replication. More specifically, the siRNA of modification is modified in the ribose 2 ' position of siRNA. Modification occurs in 2 ' of described at least one ribonucleotide of siRNA. Can utilize the combination of receptors bind part and siRNA molecule to make the required cell type of siRNA target. For example, make cholesterol be incorporated into 5 ' of siRNA molecule-end or 3 '-end obtains cholesteryl siRNA, can strengthen it to hepatocellular target-seeking. The part that is used for the receptor-mediated siRNA of target liver comprises HBsAg, LDL etc.

More particularly, siRNA is modified at least one pyrimidine, at least one purine or its combination. Yet the combination of the pyrimidine that common siRNA is all or all purine or all pyrimidines and all purine is all modified. More preferably pyrimidine is modified, and these pyrimidines are cytimidine, cytosine derivative, uracil, uracil derivative or its combination. Can also modify the ribonucleotide through selecting in siRNA at least one chain, perhaps the ribonucleotide in all two chains of siRNA all can be modified.

The nucleotides that contains the pyrimidine bases (cytimidine and uracil) of RNA can be modified with chemical method, and method is to suppress the molecule of RNA degraded or decomposition in 2 ' one of adding of ribose molecule. Available multiple distinct methods forms the pyrimidine nucleotide of 2 '-modification. This 2 ' modification is by making siRNA not affected by nuclease or it being produced the stability that resistance improves siRNA. Therefore, this 2 ' siRNA that modifies has long serum half-life, with the siRNA phase specific energy opposing degraded of unmodified. SiRNA also can be modified wholly or in part.

When chemical modification siRNA, preferably the halide chemical group is added the ribonucleotide of siRNA. In halide, fluorine is preferred molecule, but other chemical molecular outside the fluoro-, as methyl-, methoxy ethyl-and propyl group-modification also can use. But preferred the modification is that fluoro-is modified, and modifies or the modification of 2 ', 2 '-fluoro-such as 2 '-fluoro-. Therefore, in a preferred embodiment of the invention, add fluorine molecule by 2 ' carbon at the pyrimidine ribonucleotide and modify siRNA. SiRNA can be fluoridized wholly or in part. For example, only have cytidylic acid to be fluorinated. Perhaps, only have uridylate to be fluorinated, but uracil and crisp pyrimidine all can be fluorinated. In addition, only have the chain (justice or antisense strand) of siRNA to be fluorinated. Even if fluoridizing also, siRNA part 2 ' can resist nucleolysis. In addition, it is pointed out that the 2 ' siRNA that fluoridizes is nontoxic to cell, in view of fluorine chemistry normally poisonous to the organism that lives, so this is a unexpected result.

Designing siRNA of the present invention can interact with target nucleotide sequences. Most preferably this target nucleotide sequences is to wish to suppress the virulence factor of its gene expression or the sequence of pathogen. More preferably this target nucleotide sequences is the sequence in the viral genome, and this viral genome is from the RNA virus or the dna virus that are selected from lower group in addition: hepatitis C virus (HCV), hepatitis A virus, hepatitis B, Hepatitis D virus, HEV, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, HPV (HPV), super Pneumovirinae and coronavirus. Most preferred virus is SARS virus.

The siRNA that modifies can prepare in many ways, as transcribing by chemical synthesis, T7 polymerase or with the long dsrna (dsRNA) of cutting the modification that enzyme modification prepares with one of above-mentioned two kinds of methods. Cut enzyme and can be used to cut the dsRNA that contains 500-1000 the base-pair of having an appointment, the dsRNA that produces the about 21-23 of a length base-pair mixes the group. In addition, adopt a beyond thought result who cuts enzyme method to be, cut enzyme and will cut the dsRNA chain that process is modified, fluoridize the dsRNA of modification such as 2 '. Set up before this method, once thought and cut the siRNA that enzyme can not cut modification. Cutting enzyme process can use the Dicer siRNA Generation Kit available from Gene Therapy Systems (San Diego, CA) to carry out.

SiRNA (siRNA) is defined as length here and is about 10-30 nucleotides, 12-28 nucleotides is more preferably arranged, 15-25 nucleotides is more preferably arranged, 19-23 nucleotides is more preferably arranged again, the two strands of 21-23 nucleotides-or single stranded RNA is most preferably arranged. The length that is used for the siRNA of this paper is to determine with the length of a chain of RNA. For example, length is that the siRNA of 21 nucleotides (21-mer) can contain two relative RNA chains, and they are annealed together and obtain 19 base-pairs that adjoin. Two residual nucleotides of this molecule one end can not annealed together with relative chain, thereby form " jag ". Jag can be at 5 ' or the 3 ' end of dsRNA. Preferred jag is at the 3 ' end of RNA. The different double-stranded RNA of two relative chain lengths will represent with of growing in two chains. For example, think 21-aggressiveness (21-mer) with the dsRNA that the relative chain annealing of long 20 nucleotides forms at this paper by a chain of long 21 nucleotides.

Preferred siRNA of the present invention will contain 3 ' jag of 2-4 the base of having an appointment. More preferably this 3 ' jag length is 2 nucleotides. More preferably 2 contained nucleotides of 3 ' jag are uracil (U).

In one embodiment, RNA molecule provided by the invention contains the nucleotide sequence identical with the nucleotide sequence at least 80% of target virulence factor or virus. The nucleotide sequence at least 90%, 95%, 96%, 97%, 98% of preferred RNA molecule of the present invention and target preparation or virus, 99% or 100% identical.

In practice, adopt known computer program, such as Bestfit program (Wisconsin sequence analysis software bag, Unix version 8, science of heredity calculates unit, University Research Park, 575Science Drive, Madison, Wis.53711), measure any specific nucleic acid molecule whether with the nucleotide sequence at least 90%, 95%, 96%, 97%, 98% of target virulence factor or virus, 99% or 100% identical. Bestfit uses local homology's algorithm (Advances in Applied Mathematics 2:482-489 (1981)) of Smith and Waterman can find the best section of homology between two sequences. Whether for example 95% identical with reference sequences of the present invention when measure certain concrete sequence with Bestfit or any other sequence contrast program, need setup parameter, certainly, same percentage is calculated with reference nucleotide sequence total length, at this moment, 5% of homology breach as many as reference sequences nucleotides sum allow.

The invention provides the hit method of virulence factor (preferred virus) of a kind of deactivation patient, described method comprises the siRNA of the modification that gives this target virulence factor of patient's deactivation or viral effective dose. Can realize the RNA of target DNA section in the cell is disturbed by giving cell dsRNA molecule or siRNA, wherein, the nucleotide sequence of dsRNA molecule is corresponding with the nucleotide sequence of this target DNA section. Preferably being used for inducing the RNA molecule of target RNAi is siRNA.

Gene inhibition, target-seeking inhibition, sequence-specific inhibition, target-seeking RNAi or sequence-specific RNA i here are used interchangeably. In addition, it is by measuring respectively the level of downtrod protein in the cell (test cell) contain siRNA and the cell that does not contain identical siRNA (control cells) here that sequence-specific is suppressed at, and these two measured values of comparison and definite. In addition, test cell and control cells must be from same source and same animals. For example, control cells and test cell can be (but being not limited to) human liver cell and external cell culture, and perhaps they can be derived from hepatocellular carcinoma. In addition, the control cells that is used for mensuration gene inhibition level or gene amount of suppression must be measured under similar (if not identical) condition with the test cell.

Phrase " target DNA section " this paper is used for being expressed as the dna sequence dna of all or part of mRNA that needs the coding target protein that suppresses, comprises introne or extron. The DNA section also can represent usually to regulate the dna sequence dna that target protein is expressed, and includes but not limited to the promoter of target protein. In addition, the part that described DNA section can the yes or no cellular genome can be exchromosomal DNA perhaps, such as DNA.

The invention still further relates to certain viral method in the deactivation patient body, comprise the siRNA that gives the modification that patient's deactivation should the virus effective dose. The length of described siRNA preferably is about 10-30 nucleotides, more preferably 12-28 nucleotides, more preferably 15-25 nucleotides, more preferred 19-23 nucleotides, most preferably 21-23 nucleotides. 2 ' adorned 2 ' siRNA that modifies that the method is preferably used at described at least one ribonucleotide of siRNA. The method use be selected from fluoro-, methyl-, methoxy ethyl-and the siRNA of the chemical group modification of propyl group-modifications. It is preferred that fluoro-is modified, 2 '-fluoro-is modified or 2 ', 2 '-fluoro-to be modified in this also be useful and also be preferred.

Modification can be the modification in siRNA pyrimidine, purine or its combination. More preferably pyrimidine is modified, such as cytimidine, cytosine derivative, uracil, uracil derivative or its combination. In one embodiment, siRNA chain contains the nucleotides of at least one modification at least, and in another embodiment, two chains of siRNA all contain the nucleotides of at least one modification.

The virulence factor that this method is used for or pathogen, more specifically be virus, can be RNA virus or dna virus, they can be selected from lower group: hepatitis C virus (HCV), hepatitis A virus, hepatitis B, Hepatitis D virus, HEV, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, HPV (HPV), super Pneumovirinae and coronavirus. Preferred target virus is SARS virus. The siRNA:(a that the method utilization prepares by the following method) the identifying virus genome target nucleotide sequences in the SARS virus especially is with design siRNA (siRNA); (b) make through modifying the siRNA with the nucleotides that contains at least one modification. More preferably, described siRNA comprises the dsRNA molecule, the ribonucleotide acid sequence of its article one chain is corresponding with the nucleotide sequence corresponding to described viral target nucleotide sequences, the second chain contains the ribonucleotide acid sequence with described target nucleotide sequences complementation, wherein said first and second chains are the complementary strands that separate, their phase mutual crosses form described dsRNA molecule, in addition, wherein said the first chain ribonucleotide acid sequence, the second chain ribonucleotide acid sequence or the first and second chain ribonucleotide acid sequences comprise the nucleotides of at least one modification. In the method, target nucleotide sequences comprises SARS virus and copies essential conservative nucleotide sequence, and described conservative nucleotide sequence is selected from SEQ ID NO:7292, SEQ ID NO:7293, SEQ ID NO:7294, SEQ ID NO:7295, SEQ ID NO:7296, SEQ ID NO:7297, SEQ ID NO:7298, SEQ ID NO:7299, SEQ ID NO:7300 and SEQ ID NO:7301. Preferred described nucleotide sequence is selected from SEQ ID NO:7292 and SEQ ID NO:7293. More preferably, described nucleotide sequence is SEQ ID NO:7293.

The described siRNA of the application can make with the ribonucleotide of modification described here. In addition, can will mix described siRNA for the ribonucleotide of the modification of the siRNA of the inventive method by chemical synthesis or enzymatic synthesis.

The described siRNA of the application can contain or not contain 5 ' triphosphoric acid group.

The siRNA that modifies be by be selected from intravenous injection, hypodermic injection, the oral and defeated method of passing of liposome gives. The siRNA that modifies condenses in patient's the body systems such as organ, tissue or liver, intestines and stomach, respiratory tract, uterine neck or skin.

The method that the present invention also provides heterogeneous SARS virus positive cell inner virus (such as SARS virus) to copy comprises the carrier transfection SARS positive cell of expressing through the SARS specific siRNA of modifying with instructing. Cell is estimated to determine whether adorned siRNA suppresses intracellular label.

The term patient here can be animal, preferred mammal. Preferred to liking primate, comprise non-human primates and people. Term " object " and " patient " are used interchangeably.

Before can be used for, the imaginary processing method of the present invention by the object of virus infections, or have SARS virus to infect the object of tendency before being used for. In addition, method of the present invention can be used for correcting or compensate and makes the patient to the relevant cell of virus infections susceptible or physically different, and/or is used for alleviating patient's virus infections symptom, or as patient's precautionary measures.

Treatment virus infections patient's method comprises and gives this group of objects compound. Composition can represent pure compound, reagent or material here, or the mixture of two or more compounds, reagent or material. Term " preparation, material or compound " refers to protein, nucleic acid, carbohydrate, lipid, polymer or little molecule here, such as medicine.

In an embodiment of the present invention, the composition that gives object is pharmaceutical composition. In addition, but in the described pharmaceutical composition per os, intranasal, parenteral, system, in the peritonaeum, local (as by drops or transdermal route), contain and take or as mouthspray or nose spray application. Term " parenteral " represents mode of administration here, comprise in the intravenous, muscle, in the peritonaeum, breastbone is interior, subcutaneous and intra-articular injection and infusion. Pharmaceutical composition of the present invention also can contain pharmaceutically acceptable carrier.

" pharmaceutically acceptable carrier " refer to, but be not limited to, and the prescription auxiliary material of nontoxic solid, semisolid or liquid filling agent, diluent, coating material or any type is such as liposome.

Pharmaceutical composition of the present invention for parenteral injection can contain the pharmaceutically acceptable aseptic aqueous solution or non-aqueous solution, dispersion liquid, suspension or emulsion, and the aseptic powdery of rebuilding with injectable sterile solution or dispersion liquid before use. Suitable moisture or aqueous carrier, diluent, solvent or carrier do not comprise that water, ethanol, polyalcohol are (such as glycerine, propane diols, polyethylene glycol etc.), carboxymethyl cellulose and suitable mixture, vegetable oil (such as olive oil) and injectable organosilane ester such as ethyl oleate thereof. For example, can adopt coating material such as lecithin, keep granularity required in the dispersion liquid, and keep suitable flowability with surfactant.

Composition of the present invention also can contain adjuvant, such as, but not limited to, anticorrisive agent, wetting agent, emulsifying agent and dispersant. Add various antiseptics and antifungal agent and can guarantee to prevent the effect of microorganism, such as metagin, anesin, phenol, sorbic acid etc. Also can contain isotonic agent, such as sucrose, sodium chloride etc. The reagent that the adding delay absorbs such as aluminum monostearate and gelatin can delay the absorption of injectable drug preparation.

Under the certain situation, for prolonging drug effect, can slow down from absorption subcutaneous or the intramuscular injection medicine. This can realize by using water-soluble relatively poor crystal or the liquid suspension of amorphous substance. The absorptivity of medicine depends on its dissolution velocity, then can be depending on grain size and crystal form. Perhaps, can be by with medicine dissolving or be suspended in the absorption that the oiliness carrier postpones the medicament forms of parenteral administration.

Injectable long-acting form can be included in the polymer of biodegradability such as the microencapsulation material of the medicine in polylactide-PGA is made by formation. According to the ratio of medicine and polymer and the character of particles used polymer, controlled pharmacy rate of release. The example of the polymer of other biodegradability comprises poly-(ortho esters) and poly-(acid anhydrides). Also medicine can be wrapped into the liposome compatible with bodily tissue or microemulsion is made injectable durative action preparation.

Injectable formulation can be sterilized, and for example filters by the filter of holding back bacterium or adds solubilized or be dispersed in the bactericide of the aseptic solid composite form in the sterilized water or add before use other injectable sterile media.

Include, but not limited to capsule, tablet, pill, powder agent and granule for oral solid dosage forms. In this solid dosage forms; reactive compound can with at least a pharmaceutically acceptable excipient or carrier; for example natrium citricum or or Dicalcium Phosphate mix mutually; and/or a) filler or bulking agent; such as starch, lactose, sucrose, glucose, sweet mellow wine and silicic acid; b) adhesive; such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum; c) NMF, such as glycerine, d) disintegrant; such as agar, calcium carbonate, farina or tapioca, alginic acid, some silicate and sodium carbonate; e) solution is detained agent, such as paraffin, and f) sorbefacient; such as quaternary ammonium compound; g) wetting agent is such as the monostearate of acetyl-ethanol and glycerine, h) adsorbent; such as kaolin and bentonite; and i) lubricant, such as talcum, calcium stearate, dolomol, solid polyethylene glycol, NaLS, and their mixture. If capsule, tablet and pill also can contain buffer in this formulation.

Similarly solid composite also can be used as the filler of soft hard gelatin capsule, uses lactose and high molecular weight polyethylene glycol etc. as excipient.

The solid dosage forms such as tablet, lozenge capsule, pill and granule can contain dressing or shell, other coating of knowing such as casing and pharmaceutical field. They can be chosen wantonly and contain opacifier, also can be only by or the preferential composition of certain part (can choose wantonly in the slowly-releasing mode) release of active ingredients by enteron aisle. The example of operable embedding composition comprises material and the wax of polymerization.

Reactive compound also can be microencapsulation form, if suitable one or more above-mentioned excipient that also contains.

Include, but not limited to pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir for Orally administered liquid dosage form. Except reactive compound, described liquid dosage form also can contain this area inert diluent commonly used, for example water or other solvent, solubilizer and emulsifying agent, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, phenmethylol, Ergol, propane diols, 1,3-butanediol, dimethyl formamide, oils (especially cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor oil and sesame oil), glycerine, tetrahydrofurfuryl carbinol, the fatty acid ester of polyethylene glycol and anhydrosorbitol, and their mixture.

Except inert diluent, also can contain adjuvant in the Orally administered composition, such as wetting agent, emulsifying agent and suspending agent, sweetener, flavor enhancement and aromatic radical.

Also can contain suspending agent except reactive compound in the suspension, such as the different hard ester alcohol of ethoxylation, polyoxyethylated sorbitol ester and sorbitan ester, microcrystalline cellulose, inclined to one side aluminium hydroxide, bentonite, agar and bassora gum, and their mixture.

Perhaps, described composition can be pressurized or contain Compressed Gas, such as nitrogen or gas propellant. The liquefaction propulsive medium, it is preferred being actually all compositions, active component just can not be dissolved in wherein with any substantial degree like this. The composition of supercharging also can contain surfactant. Surfactant can be liquid or solid-state non-ionic surface active agent, perhaps can be solid-state anion surfactant. The preferred solid-state anion surfactant that uses sodium-salt form.

Composition of the present invention can also liposome form use. As known in the art, liposome is usually from phosphatide or other lipid material. Single or multiple lift hydration liquid crystal can be dispersed in and form liposome in the water-bearing media. Can use on any physiology that can form liposome and can accept and metabolizable nontoxic lipid. Except compound of the present invention, the composition of the present invention of liposome form can contain stabilizing agent, anticorrisive agent, excipient etc. Preferred lipid is natural and synthetic phosphatide and phosphatid ylcholine (lecithin). The method that forms liposome be known in the art (referring to, for example, Prescott compiles, Meth.Cell Biol.14:33 is with reference to following (1976)).

The technical staff who is proficient in this field should be understood that the effective dose of reagent of the present invention can determine according to experience, and can purified form or use with pharmaceutically acceptable salt, ester or prodrug forms (if having this form). Described reagent can be used as the object that is applied to needs treatment virus infections with the pharmaceutical composition of one or more pharmaceutically acceptable mixed with excipients. Be also to be understood that when giving human patients total consumption will be determined by the doctor in charge day of reagent of the present invention or composition in the reasonable scope. The particular treatment effective dose that is used for any particular patient will depend on various factors: the cell that need to reach or the type of physiological reaction and degree; The activity of used concrete reagent or composition; Employed concrete reagent and composition; Patient's age, body weight, health status, sex and diet; Administration time, method of administration, and excretion of drug speed; The treatment duration; The medicine that is used for composition or uses simultaneously with particular agent; And other similar factor of knowing of medical domain. For example, the technical staff who is proficient in this field knows to be lower than the level that obtains required curative effect required dosage and begins to give reagent, and the dosage that then raises gradually is until reach required effect.

Administration can the special mode of patient be carried out, so that the haemoconcentration with acceptable technology and the predetermined medicament of this area convention to be provided. Therefore can change patient's dosage regimen to realize the adjusting to carrying out property blood levels, this level is measured by HPLC, and rank is 50-1000ng/ml.

Those of ordinary skill in the related art will be understood that, in the situation that does not exceed the present invention and any embodiment scope thereof, can carry out other to methods and applications described here and suitably revise and improve.

The siRNA that modifies is prepared with conventional chemical synthesis at Lafayette CO. by Dharmacon. Each C in the siRNA duplex (GL2) and U are replaced by 2 '-F-U and 2 '-F-C, but except the 3 '-terminal jag dTdT.

Compare with the siRNA (siRNA) of unmodified for test, the stability of the siRNA of 2 ' chemical modification has been carried out following test. It is 80% human serum from healthy donors of 20 μ L that 4 nanogram siRNA are added volume. With this mixture at 37 ℃ of different times of cultivating 1 minute-10 days. The siRNA (2 '-F siRNA) that 2 ' fluorine is modified carries out same processing. After cultivating process finishes, mixture is placed on ice and separate by PAGE immediately, use the 32P-siRNA contrast. Compare with the siRNA of unmodified, the 2 ' siRNA that modifies is stable.

V. identify and be used for the treatment of the therapeutic activity agent that SARS virus infects

The invention provides the method for the treatment of SARS by giving mammal therapeutic activity agent (such as micromolecular compound), and the method for identifying the therapeutic activity agent (such as effective little molecule) that is used for the treatment of the SARS virus infection.

One aspect of the invention provides the method for identifying the therapeutic activity agent, and the method comprises: the cell that therapeutic activity agent contact is infected by SARS virus; (b) attenuation of mensuration SARS relevant enzyme.

One more in the specific embodiment, described therapeutic activity agent is little molecule. In other more particular embodiment, described therapeutic activity agent is nucleoside analog (for example Ribavirin). In other more particular embodiment, described little molecule is that SMIP or peptide para-immunity are regulated compound. In other more particular embodiment, described therapeutic activity agent is class peptide, oligopeptides or polypeptide. In another embodiment, the SARS relevant enzyme is SARS protease. In another embodiment, the SARS relevant enzyme is the SARS polymerase. Again in other embodiments, the SARS relevant enzyme is a kind of kinases. Again in other embodiments, the SARS relevant enzyme is a kind of protease. Furin inhibitor peptidyl chloromethyl ketone can stop the metainfective cell-Fusion of Cells of MHV (de Haan etc., (2004) J Virol), and this provides guidance for the SARS treatment.

The present invention includes a kind of mensuration based on cell that can be used to the therapeutic activity agent of screening and identification treatment SARS virus infection. Therapeutic activity agent of the present invention comprises inhibition, prevents or reduces the reagent that SARS virus is copied. The cell (for example VERO cell) that available SARS virus infections is cultivated is also estimated possible antiviral compound this reagent is identified in the impact that SARS virus infects. To measure possible antiviral compound be well known in the art to the assay method of the effect of virus replication and can be based on many kinds of parameters.

Mensuration based on cell can be used for high flux screening to identify therapeutical active compound from the chemicals storehouse of containing potential antiviral compound. Be applicable to therapeutical active compound of the present invention and can suppress virus at the complete necessary any SARS virus target of time multiplexed cell system. The effect of therapeutic agent (compound suppresses or deactivation target virus or cell, the ability that causes the culture inner virus to reduce) can be measured by vigor and/or the propagation of estimating survivaling cell in the cell culture that is infected by SARS virus.

Many methods that is used for measuring cell viability known in the art are such as the determination method of the enzyme, protein, nucleotides triphosphoric acid (such as ATP), nucleic acid (such as mRNA (for example GAPDH) or the rRNA sequence of host cell) or cell metabolite such as MTT or the MTS that measure cell. In addition, the DNA of available fluorescent dye (comprising for example HSV paper) or non-fluorescent dye (for example propidium diiodide) or mark measures cell viability and/or propagation phenomenon.

Perhaps, can determine by the amount of virus or viral product in the direct measurement culture effect of compound or specimen. The method of measuring the amount of virus, viral genome or viral product comprises: PCR, RT-PCR, TMA, the reporter protein with fluorescence or the characteristics of luminescence or enzyme function (such as luciferase, alkaline phosphatase, GFP), or can be by the protein of antibody test (such as EGF), they can mix viral genome before cell culture is infected. In addition, viral product such as virus protein can be measured by ELISA or enzymatic activity. The method of identifying virus polynucleotides, virus protein and virus protein specific antibody is narrated hereinbefore.

Potential antiviral compound is used for mensuration based on cell with the concentration of about 10 μ M, and identifies the type of compounds with therapeutic efficiency by the selected parameter of measurement (such as cell viability/propagation or virus or viral genome is viral or the viral product in non-viral source). In case think that compound has activity, they can be synthesized and be simulated again. From certified compound, many analogs and novel compound in synthetic, the biological propagation of continuous the best and modeling circulation, have been synthesized so that exemplary configuration is best, until activity in vivo is illustrated and is optimised.

The cell that is applicable to this mensuration comprises the cell of above-mentioned suitable production vaccine. Preferred described cell is African green monkey kidney cell (Vero) cell. HELF or normal person's diploid fibroblast also can be used for the present invention.

In one embodiment, the present invention includes a kind of cytopathogenicity determination method based on fluorescence and measure potential antiviral compound to the impact based on the mensuration of cell. An example of this cytopathogenicity determination method based on fluorescence is as described below.

The every hole 1 * 10 of microtiter plate (MTP)⁴The Vero cell infects with the SARS virus of determining amount, SARS virus has following best MOI scope: 5-10,10-25,25-50,50-100,100-500 or 500-1000PFU, culture medium (replenishing the M199 culture medium of 5%FCS, 2mM glutamine, 100IU/ml penicillin and 100 μ g/ml streptomysins) cumulative volume is 200 μ l, wherein exist or do not have potential antiviral compound, and at 37 ℃, 5%CO₂Cultivated at least 1,2,3,4,5,6 or 7 day. Then each hole of MTP adds the PBS that 200 μ l contain 10 μ g/ml fluorescein diacetates with PBS (200 μ l) washing. Room temperature was cultivated after 45 minutes, measured fluorescence with 485nm excitation wavelength and 538nm emission wavelength. Drafting is determined IC as the non-linear figure of the antiviral activity of drug concentration function₅₀Value.

Other mensuration based on cell is known in the art, comprising GFO detection method and Luc detection method. In addition, also can measure cell viability with the Promega kit that obtains by commerce.

In one embodiment, the present invention includes the method for measuring potential antiviral compound effect, the method uses RT-PCR to detect the level of SARS virus RNA in based on the mensuration of cell. Use the method for RT-PCR known in the art. An example of this assay method is as described below.

With 5 * 10⁶The Vero cell is inoculated in tissue culture. To contain the flask of cell at 37 ℃, 5%CO₂Cultivation is spent the night. When existing and do not have potential antiviral compound, infect (m.o.i.=1) cell with SARS virus. Randomly, can be with potential compound treatment cell before infecting. Which kind of situation has all been carried out suitable control cells and has been measured.

Infect the RNA of rear 2 hours (UL54), 12 hours (UL8) and 16 hours infected cells of (UL13) purifying, quantitative assay (Qiagen) RNA purity (Rneasy kit; 40 μ l wash-outs) (260nm absorption). According to Superscript II scheme (Invitrogen), with Auele Specific Primer (2pmol, with above-mentioned primer to one of) RNA (2 μ g) reverse transcription is become cDNA. By pcr amplification reverse transcription equal portions reactant (2 μ l). Suitable target SARS genetic fragment, i.e. the gene of codase, by PCR (the Taq polymerase, Stratagene) (UL54 and UL8:94 ℃ thermal starting, 3 minutes are taken turns in amplification 30; 94 ℃ of sex change, 1 minute, 55 ℃ of annealing, 1 minute; 72 ℃ of polymerizations, 1 minute. UL13:94 ℃ of thermal starting, 3 minutes; 94 ℃ of sex change, 1 minute, 60 ℃ of annealing, 1 minute; 72 ℃ of polymerizations, 1 minute), reaction volume is 100 μ l, contains above-mentioned suitable oligonucleotides, every kind of 0.1nmol. 20-30 wheel PCR cyclic amplification 8 μ l aliquot samples (swimming lane 2-12) are analyzed with 2% Ago-Gel (Invitrogen) according to the explanation of manufacturer.

Mensuration based on cell of the present invention can be chosen variant or the derivative of the wild type SARS virus of using in people and/or animal model (such as mouse, non-human primates etc.) virulence reduction or attenuation wantonly. In screening technique, use this attenuation SARS virus can reduce with security-related problem and can reduce pathogenic vigilant of SARS virus, and can eliminate or reduce and measure and carry out the demand that high level is controlled during screening compounds.

The present invention includes a kind of assay method based on enzyme that can be used to the therapeutic activity agent of screening and identification treatment SARS virus infection.

One embodiment of the invention are a kind of assay methods that may further comprise the steps: the SARS protease that makes known quantity in the solution with contain the peptide of detectable label with SARS protease cutting site and contact, wherein, the SARS proteinase activity is monitored by the label density of measuring cleaved products.

At one more in the specific embodiment, a kind of method of the SARS of mensuration protease is provided, the method comprises to be made the sample solution that contains SARS protease and contains fluorescence donor, fluorescence quencher and the peptide of SARS protein cleavage site and contact, after being cut, described peptide can detect by fluorescence photometer, wherein, the SARS proteinase activity in the sample is determined by the fluorescence volume that fluorescence photometer detects.

Can use based on the inhibiting determination method of direct measurement SARS protease and screen the SRAR therapeutic agent. The protein that is used for this mensuration can separated and purifying such as 3C sample protease and papain sample protease, as with as described in the Publication about Document: Seybert etc., J.Gen.Virol., 78:71-75,1997, Ziebuhr etc., Adv.Exp.Med. Biol., 440:115-120,1998, Sims etc., Adv.Exp.Med.Biol.440:129-134,1998, Ziebuhr etc., J.Virol., 73:177-185,1999, Teng etc., J.Virol., 73:2658-2666,1999, Herold etc., J.Biol.Chem.274:14918-14925,1999; With Ziebuhr etc., J.Biol. Chem.276:33220-33232,2001. In addition, embodiment 30 has described a kind of new method with column chromatography purification SARS protease. Embodiment 31 has described FRET (FRET) determination method to measure the SARS proteinase activity. Embodiment 31 shows, is applicable to high flux screening based on determination method such as the FRET determination method of protease, and can be used to screen candidate's antiviral compound. When having the SARS protease inhibitor compound, the result of protease assay method shows, compares with the negative control that does not contain test compounds on the non-inhibity control compound, and the fluorescence volume of a certain preset time reduces. This method will may further comprise the steps: the test solution that contains SARS protease (a) is provided; (b) in test solution, add test compounds; (c) in test solution, add the SARS protease substrate; (d) proteolytic activity of measurement test solution. In preferred embodiments, proteolytic activity is the fluorescence measurement by the fluorogen product made by the enzymatic activity of SARS protease.

Attenuation SARS virus variant comprises one or more genomic modifications or sudden change (for example, replace, lack, insert) usually in code area or the code area of protein. The object lesson of attenuation mutant comprises, for example, the genetic modification in any nonstructural protein gene outside 5 '-terminal noncoding region, targeting sequencing, intergenic region, 3 '-terminal noncoding region, ORF 1a, ORF 1b, S gene, E gene, M gene, N gene or the ORF 1a/1b district. Preferred attenuation sudden change is positioned at SARS virus structural proteins (for example, furcella (S)), protease or Polymerase Structure territory or non-coding sequence (for example, 5 '-terminal noncoding region, intergenic sequence). In addition, can in spike protein, mix or delete cleavage site (referring to for example, Gombold etc., J.Virol.67:4504-4512,1993; Bos etc., Virology 214:453-463,1995), such modification also is useful (for example, being used for vaccine) for the expression of optimum combination spike protein antigen.

As described herein, used several different methods to obtain the SARS virus variant of attenuation. These methods are included in continuous passage SARS virus in the cultured cell (for example, mammalian cell cultures is such as fetal rhesus kidney cell or VERO cell) until confirm that SARS virus is attenuated. The continuous propagation of virus can be carried out under tissue culture goes down to posterity any temperature that attenuation can occur, and can be in conjunction with one or more other mutagenesis steps (such as mutagenesis). The go down to posterity attenuation phenotype of SARS virus mutant of rear acquisition of one or many cell culture is that the technical staff who is proficient in this field is not difficult to measure. Attenuation refers to that here the virulence of SARS virus in human subjects reduces. The virus replication level that shows as the evidence of attenuation reduces or virulence reduction in animal model.

Other method of making the attenuation SARS virus is included in time Jia Wendu cell culture and goes down to posterity viral (cold going down to posterity), and by random mutagenesis (for example carrying out mutagenesis with 5 FU 5 fluorouracil) or with direct mutagenesis the attenuation mutant is introduced the SARS virus genome. The manufacturing of attenuation RSV vaccine and generation (its method is applicable to SARS virus usually) are described in, and for example, EP 0640128, U.S. Patent No. 6,284,254, U.S. Patent No. 5,922,326, U.S. Patent No. 5,882,651.

But obtain the required at least part of condition that adopts that depends on of passage number of attenuated virus of the immunity inoculation of safety. Regularly detect virulence and the immunocompetence of SARS virus culture in animal (such as mouse, primate) and can determine easily the concrete parameter that makes up of particular organization's culture and temperature.

In another embodiment, be used for screening antiviral compound based on the mensuration of cell based on being not the reading that the gene outcome (for example reporter product) from SARS virus is expressed. Be particularly suitable for gene outcome of the present invention and include but not limited to used those in the said determination.

For obtaining this class reading, the interested gene (GOI) of described gene reporter gene product of encoding must be impregnated in the SARS virus genome that copying or from the genomic construction of SARS virus (for example, SARS virus replicon, the damaged interference of SARS virus (DI) RNA). Figure 13 shows reporter gene and mixes the genomic position of SARS virus. Preferred interested allos reporter gene is inserted between as shown in figure 13 the existing SARS virus gene. For example, GOI can be inserted into immediately following the position (for example, ORF 1b, S, E, M, N) after the SARS virus gene terminator codon. Insertion should be positioned and interrupt minimum so that the SARS virus gene mRNA is transcribed. GOI also can be used as frame interior " fusant " and insert with existing SARS virus gene, can keep so the enough functions of GOI to detect. Express optimum for making, also other SARS virus intergenic sequence can be built into (for example, SEQ ID NO:7388 contains or do not contain other flank SARS virus sequence) the GOI position before of insertion.

GOI is mixed SARS virus can have the technical staff who is proficient in this field to finish with various technology. For example, preferred method is target RNA restructuring, its advantage be in cell, to recombinate coronavirus RNA (referring to such as Fischer etc., J.Virol.71:5148-5160,1997; Koljesar etc., J.Vet.Sci.2:149-157,2001). Made and (for example contained GOI and flank SARS virus sequence, intergenic sequence) required configuration (for example, the cDNA of SARS virus defective interfering RNA) construction, thereby can be in eukaryotic or at external direct transcribe rna, and it be transfected into the permissive cell that is also infected by SARS virus. Identify the recombinant virus that contains GOI according to the expression of GOI coded markings.

Perhaps, GOI is mixed SARS virus can be finished by the technical staff who is proficient in this field, method is at first to assemble the full-length cDNA of SARS virus, and it can be used in vivo (for example from the rna plymerase ii promoter) or external (for example from phage promoter) and makes the infectious RNA transcript. Although genome length is relatively long, the technical staff who is proficient in this field has been not difficult to obtain the assemblage of this class full length cDNA clone (referring to for example now with the genome sequence of standard molecular biology and reverse Genetics Technique and SARS virus, Thiel etc., J.Gen.Virol., 82:1273-1281,2001; Almazan etc., Proc.Natl.Acad.Sci.USA 97:5516-5521,2000; Thiel etc., (2003) J Gen Virol 82:1273-1281; Yount etc., (2003) PNAS USA 100:12995-13000). Available various technology is inserted total length SARS virus genome cDNA with allos GOI, for example, connects into natural or synthetic restriction site, PCR (for example overlapping PCR) and restructuring.

Can also carry out Antiviral screening with the less SARS virus recombinant that contains gene of interest, still, need further to modify the cytopathy (for example CPE) at utmost to reduce or eliminate virus induction. The acellular pathology derivative of SARS virus can be by the technical staff who is proficient in this field acquisition that ins all sorts of ways. For example, but selected marker (for example drug resistance mark) can be mixed the SARS virus genome to make above-mentioned infectious virus (referring to for example, Perri etc., J.Virol., 74:9802-9807,2000) as GOI. Then the infectious SARS virus or the infectious gene group RNA/cDNA that contain GOI can be used to infection/transfectional cell (for example VERO), can carry out mutagenesis before or not carry out mutagenesis, then select the cell of infection with suitable back-and-forth method. But only having those to contain has simultaneously the cell of SARS virus that selected marker and one or more make virus be the sudden change of acellular pathology and could survive in selection course and grow. Detect easily copying of these intracellular reactive SARS viruses with various detection techniques (for example PCR, Northern trace), this cell can be used as the substrate based on the Screening test of cell. Cause the sudden change of required acellular pathology SARS virus phenotype can comprise that nucleotides replaces, deletes or adds, they can occur in gene coding region or noncoding region (for example, 5 ' or 3 '-terminal noncoding region, intergenic region, ORF1a, ORF1b, protease domain, Polymerase Structure territory). By changing with the exchange of the sequence of wild type (such as parental generation) SARS virus and the phenotype that shows, and suitable genome area is checked order, be not difficult to identify this sudden change. The similar sudden change that can reduce or eliminate the cell pathology effect also can be used in the replicon carrier that SARS virus derives, by carrying out similarly directly screening with SARS virus replication, or come the specificity construction replicon according to the sudden change of identifying in the above-mentioned infectious SARS virus chapters and sections. In addition, this mutant can be used as the basis of other local described attenuation SARS virus derivative of the application.

Perhaps, except carry out the Screening test based on cell with infectious SARS virus or derivatives thereof, can make up and use breeding unsoundness type " replicon ". This replicon keeps all proteins coded sequence and rna replicon reaches at the necessary cis replication sequence of cell inner expression, but the required sequence of one or more packing offspring SARS viruses or gene have been deleted (referring to such as Curtis etc., J.Virol., 76:1422-1434,2002). Figure 14 has described the representative example of SARS virus replicon of the present invention. For example produced a kind of SARS virus cDNA construction, this construction shortage one or more (or owning) structural protein matter encoding gene, the SARS viral gene that wherein lacks is replaced by GOI, keep to express simultaneously GOI necessary all transcribe signal. What operability was connected in the sub-cDNA construction of SARS virus replication is rna polymerase promoter, and it can be used in vivo (for example, rna plymerase ii promoter) or external (for example, phage promoter) transcribes replicon rna. Can the SARS replicon be introduced permissive cell as RNA or DNA (depending on selected promoter) by transfection, and estimate antiviral compound with the cell of transfection. So that replicon does not have cytopathic effect (seeing above) to cell, just need to when measuring, all not carry out nucleic acid transfection by mixing one or more sudden changes at every turn.

Perhaps, the SARS virus replicon can be packed into can infection cell virus-like particle, and do not need the transfected with core acid molecule. Requirement to the replicon packing is that the substantive SARS virus gene function (for example one or more structural proteins) of deleting from replicon provides with trans within containing the cell of this replicon. The technical staff who is proficient in this field can in all sorts of ways to pack replicon rna (referring to for example, Curtis etc., the same: Ortego etc., J.Virol., 76:11518-11529,2002). For example, can use the clone of the stable conversion of the required SARS virus gene function of composing type or inducible expression. Perhaps, required SARS virus gene function can be expressed by the viral vectors that is impregnated in the cell that contains replicon. Perhaps, contain the RNA that damaged interference (DI) SARS virus of required gene derives and to be impregnated in the cell that contains replicon. This DI construction for the replicon function that remedies forfeiture is commonly referred to damaged complementary RNA or damaged auxiliary (defective helper).

To the DI RNA that derives based on the SARS virus of the useful another kind of configuration utilization coding GOI of the Antiviral screening determination method of cell of the present invention (referring to such as Stirrups etc., J.Gen.Virol., 81:1687-1698,2000; Liao etc., Virology 208:319-327,1995). With SARS DI, as the cDNA that is connected with the rna plymerase ii promoter or as the RNA of in-vitro transcription, also import in the permissive cell that is infected by SARS and be able in mensuration, obtain the reading of GOI reporter gene product.

Being used for the system based on replicon of Rapid identification coronavirus replicase inhibitor is described in Hertzig etc., (2004) J Gen Virol DOI 10.1099/vir/0/80044-0. In brief, but this system adopts stable maintenance acellular pathology in eukaryotic can select replicon rna. The reporter gene expression of this replicon rna mediation can be used as the coronavirus never mark, the simultaneously expression of reporter gene can be used to the inhibitory action in the vitro detection test compounds, just can high flux screening replicase inhibitor thereby need not to cultivate infectious virus. Preferred replicon rna contains neomycin resistance gene in rdrp gene, the reporter gene in its downstream (for example GFP) can be expressed by the sub-gene group mRAN of replicase mediation is synthetic.

VI. treat composition and the method for SARs virus infections

The present invention relates to treat and/or prevent composition and the method for SARS. The present invention also comprises the method that treats and/or prevents SARS by at least a antiviral compound for the treatment of effective dose, and these compounds are described in table 1 and the table 2 listed United States Patent (USP) and disclosed international patent application. In an embodiment of the method, described antiviral compound is little molecule. In another embodiment, described antiviral compound is protease inhibitors. Again in one embodiment, described antiviral protease inhibitors is 3C sample protease inhibitors and/or papain sample protease inhibitors. Lopinavir/Ritonavir (Kaletra) protease inhibitors and Ribavirin therapeutic alliance demonstrate good clinical effectiveness (Chu etc., (2004) Thorax 59:252-256). In another embodiment, described antiviral compound is the RNA-directed RNA polymerase inhibitor. In another embodiment, the first antiviral compound protease inhibitors is to use with the second antiviral compound RNA-directed RNA polymerase inhibitor. The present invention also provides and at least a antiviral compound (for example from the antiviral compound of narrating in table 1 and the listed document of table 2) combined administration steroid class antiphlogistic. Steroids and Ribavirin combined therapy have been described in Fujii etc., other 10:1-7 of (2004) J Infect Chem. Cortin and Alfacon-1 combined therapy also be in the news (Loutfy etc., (2003) JAMA 290:3222-3228).

The present invention also provides the method that treats and/or prevents SARS by at least a antiviral compound that sucks the administering therapeutic effective dose, and these compounds are described in table 1 and the table 2 listed United States Patent (USP) and disclosed international patent application. On the other hand, described antiviral compound can with SMIP, SMIS or other immunomodulatory compounds (such as in table 32 and the table 33 those) combined administration. In an embodiment of the method, described antiviral compound is little molecule. In another embodiment, described antiviral compound is protease inhibitors. Again in one embodiment, described antiviral protease inhibitors is 3C sample protease inhibitors and/or papain sample protease inhibitors. In another embodiment, described antiviral compound is the RNA-directed RNA polymerase inhibitor. In another embodiment, the first antiviral compound protease inhibitors is to use with the second antiviral compound RNA-directed RNA polymerase inhibitor. The present invention also provides and at least a antiviral compound (for example from the antiviral compound of narrating in table 1 and the listed document of table 2) combined administration steroid class antiphlogistic. Described steroid class antiphlogistic can use to reach partial result by suction, perhaps for example realizes the systemic Absorption administration by oral or intravenous route.

The method that the present invention also provides a kind of SARS for the treatment of to infect, comprise use separately or with the antiviral compound combination or with the little molecular immune reinforcing agent of SARS vaccine combined administration (SMIP) compound. Again in one embodiment, described SMIP is listed compound in compound described here or the table 32.

The method that the present invention also provides a kind of SARS for the treatment of to infect, comprise use separately or with a kind of immunosuppressant compounds of antiviral compound combined administration, optional micromolecular inhibitor (SMIS) compound. Again in one embodiment, described immunosuppressant compounds is as described herein or be listed in the table 33.

The present invention also provides can affect the peptide of patient's inflammatory reaction para-immunity adjusting composition, comprises oligopeptides and polypeptide. In one embodiment, described peptide para-immunity is regulated composition can stimulate people's cell to make cell factor. In another embodiment, described peptide para-immunity adjusting composition can reduce the cytokine levels in the human body. The example that preferred peptide para-immunity is regulated composition comprises those that list in the table 35, and TGF β 2, TGF β 1, TGF β 3, thymopeptide-5 (TP5), β-mercapto propionyl-arginyl-lysyl-aspartoyl-valyl-tyrosyl-cysteine acid amides, colostrokinin, lactoferrin (LF), Cyclolinopeptide A (CLA) and tuftsin (TKPR). Peptide para-immunity adjusting composition of the present invention can use separately or be used in combination treatment SARS with the preferred antiviral compound of other preparation.

The present invention also provide a kind of for the consumer to treat and/or prevent the medicine box of SARS. Be equipped with in this medicine box: a) contain at least a antiviral, SMIP, the SMIS for the treatment of effective dose or other immunomodulatory compounds (from describe in the listed United States Patent (USP) of table 1, table 2, table 34 and table 35 and the disclosed international patent application those) and the pharmaceutical composition of pharmaceutically acceptable carrier, carrier or diluent; B) hold the container of described pharmaceutical composition; And optional comprising, c) explanation that treats and/or prevents the method for SARS with this pharmaceutical composition is described. Described medicine box can be chosen the compound that contains multiple treatment SARS wantonly, and wherein, described antiviral compound is selected from 3C sample protease inhibitors and papain sample protease inhibitors. Again in one embodiment, the antiviral compound that contains of described medicine box is the RNA-directed RNA polymerase inhibitor. When this medicine box contains that more than one are antiviral, when SMIP, SMIS or other immunomodulatory compounds, but contained compound optional combination becomes a kind of pharmaceutical composition in this medicine box.

The present invention provides other immunomodulatory compounds of describing at least a antiviral, SMIP, SMIS or the listed United States Patent (USP) of table 1, table 2, table 34 and table 35 and the disclosed international patent application to be used for the treatment of or to prevent application in the medicine of SARS in manufacturing on the other hand.

Another aspect of the present invention provides at least a SMIP compound or at least a immunosuppressant compounds or at least a SMIS compound to be used for the treatment of or to prevent application in the medicine of SARS in manufacturing. Preferred SMIP, immunodepressant and SMIS compound are as described herein.

Except as otherwise noted, following term will be for the application's VI part: " composition and the method for the treatment of SARs virus infections ", and its implication is as follows:

" restriction ", " processing " and " treatment " here are used interchangeably, and comprise prophylactic treatment (for example prophylactic) and the property alleviated treatment, or the effect of prophylactic treatment or the property alleviated treatment is provided. This term comprises and delays SARS symptom development and/or alleviate the order of severity that this type of symptom that will occur will occur or estimate after infecting SARS virus. This term also comprises to be alleviated existing SARS symptom, prevent other symptom to occur, alleviate or prevents the potential metabolic factor of symptom.

Representative compositions of the present invention and method comprise: eliminate or reduce the viral load of SARS virus in vertebrate (the comprising the people) body, eliminate or alleviate the symptom relevant with SARS, and the reduction incidence of disease relevant with SARS. In SARS patient group, use the compositions and methods of the invention to cause the high mortality relevant with SARS to reduce.

Can treat SARS virus infection and the symptom relevant with SARS by using composition of the present invention. But composition systemic administration of the present invention. For the whole body administration, can according to conventional methods compound described here be made the form that (for example, oral or rectum) carried in suitable parenteral (for example, in intravenous, subcutaneous, the muscle, in the peritonaeum, in the nose or transdermal) or the intestines. Intravenous administration can or be inculcated within the long term continuously by a series of injections. Can carry out at the certain hour interval by injection or other discrete administration that distributes, this interval can be from once in a week to once a day or three times or more. Perhaps, composition described here can be used (the rest may be inferred for applying said compositions, then drug withdrawal, and then applying said compositions) in a looping fashion. Treatment will continue until obtain results needed.

" object " is the vertebrate that needs with composition of the present invention, method and kit treatment, comprises the people. Unless sexually do not mentionlet alone brightly, term " object " comprises male and female.

The combination of " using altogether " multiple antiviral compound represents that these components can be used as a kind of composition or its part is used together with the form of single formulation. " use altogether " and also comprise and use separately multiple antiviral compound, but as the part of a kind for the treatment of procedure or scheme. " use altogether " and also comprise and use multiple other reagent, for example oligopeptides, polypeptide, peptide para-immunity conditioning agent, nucleic acid, antibody or vaccine, wherein said compound or reagent be use separately but as the part of a kind for the treatment of procedure or scheme. These components needn't be used simultaneously, although also can do so if necessary. " use altogether " that also to be included in different time individually dosed with any order. For example, the patient can also use one or more other components with one or more components in the morning at night.

" antiviral compound " here represents such as the listed United States Patent (USP) of table 1 and table 2 and the antiviral compound described in the disclosed international patent application. The United States Patent (USP) that table 1, table 2 and table 35 are listed and disclosed international patent application are included into this paper as a reference. In one embodiment, described antiviral compound is RNA-directed RNA polymerase. In other preferred embodiment, described antiviral compound is 3C sample protease inhibitors or papain sample protease inhibitors. Described antiviral compound can acid or the form of soluble alkali metal salts or alkali salt use.

The exact dose of antiviral compound will change according to dosage regimen, and seriousness and other relevant medical and the physical factors of age, body weight, sex and the symptom of object, illness to be treated depended in the selection of the oral usefulness of specific antiviral compound. Therefore, accurate medicine effective quantity can't further be specialized, and can be convenient definite by nurse or clinician.

Usually, can select the amount of suitable antiviral compound to load with the SARS virus that reduces object, and/or the reduction symptom relevant with SARS. For human, effective oral dose of antiviral compound is about every kg body weight 1.5-6000 μ g every day usually, is preferably the about 10-2000 μ of every kg body weight every day g.

One of ordinary skill in the art will be understood, some antiviral, SMIP, SMIS and immunomodulatory compounds that comprises 3C sample protease inhibitors, papain sample protease inhibitors and RNA-directed RNA polymerase inhibitor of the present invention will contain one or more to be the atom of specific spatial chemistry, tautomerism or geometric configuration, and this has just produced stereoisomer, dynamic isomer and configurational isomer. All these isomers and composition thereof all are included within the present invention when activity is arranged. In the crystal of antiviral compound of the present invention and amorphous form are also included within, also comprise hydrate, solvate and the isomorphous body of antiviral compound of the present invention.

SMIP compound of the present invention comprises the compound of describing in the disclosed United States Patent(USP) Nos. 4,547,511 and 4,738,971, has following general formula (a):

Heterocyclic radical

Be used for treating the disease that the reagent that strengthens cell-mediated immunity is responded.

Immunostimulatory oligonucleotide and polynucleotides are described in PCT WO 98/55495 and PCT WO 98/16247. The adjuvant that U.S. Patent application No.2002/0164341 describes comprises unmethylated CpG dinucleotides (CpG ODN) and non-Nuclec acid adjuvants. U.S. Patent application No.2002/0197269 has described the composition that contains antigen, antigenicity CpG-ODN and polycationic polymer.

In addition, disclosed United States Patent(USP) Nos. 4,689,338,5,389,640,5,268,376,4,929,624,5,266,575,5,352,784,5,494,916,5,482,936,5,346,905,5,395,937,5,238,944,5,525,612, WO99/29693 and U.S.Ser.No.09/361,544 have disclosed the compound of general formula (b):

Be used as " immune response dressing agent ".

Other compound with SMIP and antiviral activity is described in the U.S. Patent application that is entitled as " as the thiosemicarbazones of antivirotic and immunopotentiator " (Thiosemicarbazones as Anti-Virals and Immunopotentiators) of submitting on December 29th, 2003, the files of this application are numbered PP19814.004US, and these compounds have following structure:

The compound of formula c:

Wherein: there is not or is selected from the heteroaryl of heterocyclic radical, heteroaryl or replacement of aryl, heterocyclic radical, the replacement of cycloalkyl, aryl, the replacement of alkyl, cycloalkyl, the replacement of alkyl, replacement in E;

There is not or be selected from thiazolinyl, alkoxyl, alkyl amino, aminoalkyl, heterocyclic radical, carbocylic radical or the carbonyl of oxygen, amino, thiazolinyl, replacement in L;

There is not or is selected from the heteroaryl of heterocyclic radical, heteroaryl or replacement of aryl, heterocyclic radical, the replacement of cycloalkyl, aryl, the replacement of cycloalkyl, replacement in W;

There is not or be selected from thiazolinyl, alkoxyl, alkyl amino, aminoalkyl, heterocyclic radical, carbocylic radical or the carbonyl of oxygen, amino, thiazolinyl, replacement in X;

Y is selected from the heteroaryl of heterocyclic radical, heteroaryl or replacement of aryl, heterocyclic radical, the replacement of cycloalkyl, aryl, the replacement of cycloalkyl, replacement;

There is not or be selected from alkyl or optional heterocyclic radical, amino, alkyl amino, the dialkyl amido that replaces of F, Cl, Br, I, nitro, alkyl, replacement in Y ';

Y " there are not or are selected from alkyl or optional heterocyclic radical, amino, alkyl amino, a dialkyl amido that replaces of F, Cl, Br, I, nitro, alkyl, replacement;

R ' is the alkyl of H, alkyl or replacement;

R " be H, or

R ' and R " form together a heterocycle;

Z and Z ' independently are selected from hydrogen, alkyl, the alkyl that replaces, aryl, the aryl that replaces, aryl alkyl, the aryl alkyl that replaces, heteroaryl, the heteroaryl that replaces, heteroaryl alkyl, the heteroaryl alkyl that replaces, alkoxyl, the alkoxyl that replaces, amino carbonyl, alkoxy carbonyl, the carboxyl sulfonyl, mesyl, replace or unsubstituted alkyl-carbonyl, aryl carbonyl, aromatic alkyl carbonyl, the heteroaryl carbonyl, the heteroarylalkyl carbonyl, alkyl carbonyl oxy, aryl-carbonyl oxygen, aralkyl carbonyl oxygen base, heteroaryl carbonyl oxygen base, heteroarylalkyl carbonyl oxygen base, alkyl amino carbonyl oxy, arylamino carbonyl oxygen base, formoxyl, lower alkylcarbonyl, elementary alkoxy carbonyl, amino carbonyl, aminoaryl, alkyl sulphonyl, sulfonamido, aminoalkoxy, alkyl amino, heteroaryl amino, alkyl-carbonyl-amino, alkyl amino-carbonyl is amino, aromatic yl aminocarbonyl is amino, aromatic alkyl carbonyl is amino, the heteroaryl carbonylamino, aryl-amino-carbonyl, the ring amidino groups, cycloalkyl, cyclo-imino, aryl sulfonyl or aromatic yl sodium sulfonamido; Or

Z and Z ' form together can choose substituted heterocycle wantonly,

And tautomeride and pharmaceutically acceptable salt, ester or prodrug.

The U.S. Patent application 1O/762873 that is entitled as " the couroupitine A compound that is used for strengthening immunity " (Use of Tryptanthrin Compounds for Immune Potentiation) that other SMIP compound is described in hereinafter and submitted on January 21st, 2004, wherein disclosed the general embodiment of the compound of formula (d) expression:

Wherein,

A, B, C, D, E, F, G and H independently are selected from carbon and nitrogen, or A and B and/or C and D can represent nitrogen or sulphur together;

R ₁、R ₂、R ₃、R ₄、R ₈And R₁₀Independently be selected from thiazolinyl, amino, (alkyl of replacement) (alkyl) amino, imino group, junior alkyl halides, hydroxyl, alkoxyl, the replacement of heterocyclic radical, the replacement of alkyl, cycloalkyl, heterocyclic radical, alkyl heterocyclic, the replacement of hydrogen, halogen, low alkyl group, alkyl, replacement alkoxyl, hydroxyl alkylthio group, nitro, alkyl sulphonyl, N-alkyl sulfonamide, aryl alkyl, aryl alkyl aryl, aryl aryl, aryloxy group, arylamino, acyl amino, acyloxy amino, alkyl amino acyl amino, alkyl amino sulfonyl amino, alkyl amino, alkenyl amino, dialkyl amido, alkoxyalkyl amino, alkoxyalkyl heterocyclic radical, sulfydryl alkoxyalkyl, cyano group, formoxyl ,-COOR₁₁(R wherein₁₁Hydrogen, low alkyl group, aryl, heterocyclic radical, monose or disaccharides) or-CONR₁₂R ₁₃(R wherein₁₂And R₁₃Independently be selected from hydrogen, low alkyl group, aryl, heterocyclic radical, carbohydrate, peptide or amino acid residue); Or R₂And R₃Form together a hexa-atomic aromatic rings;

R ₇And R₉Independently be selected from heterocyclic radical or the heterocyclic radical alkyl of hydrogen, halogen, low alkyl group, junior alkyl halides, cycloalkyl, heterocyclic radical, replacement; With

R ₁、R ₂、R ₃、R ₄、R ₇、R ₈、R ₉And R₁₀When being sulphur or two key nitrogen, the annular atoms of their connections do not exist; Or its pharmaceutically acceptable salt, ester or prodrug,

Condition is, when A, B, C, D, E, F and H are carbon, and R₁、R ₂、R ₃、R ₄、R ₇、R ₈、R ₉And R₁₀Not hydrogen entirely.

In one embodiment, the compound of formula (I) has backbone structure, and wherein, D is nitrogen, and A-C and E-H are carbon.

In one embodiment, when D is carbon, R at least₁-R ₄And R₇-R ₁₀One of or two be not hydrogen.

In one embodiment, R₁-R ₄And R₈And R₁₀Independently be selected from least two in the lower group of group: the heterocyclic radical of hydrogen, halogen, low alkyl group, cycloalkyl, heterocyclic radical, replacement, alkyl heterocyclic, amino, imino group, junior alkyl halides, alkoxyl, nitro, alkyl sulphonyl, aryl alkyl, aryl alkyl aryl, aryl aryl, aryloxy group, arylamino, acyl amino, acyloxy amino, alkyl amino acyl amino, alkyl amino sulfonyl amino, alkyl amino, alkenyl amino, dialkyl amido, alkoxyalkyl amino, alkoxyalkyl heterocyclic radical, sulfydryl alkoxyalkyl, cyano group, formoxyl ,-COOR₁₁(R wherein₁₁Hydrogen, low alkyl group, aryl, heterocyclic radical, monose or disaccharides) and-CONR₁₂R ₁₃(R wherein₁₂And R₁₃Independently be selected from hydrogen, low alkyl group, aryl, heterocyclic radical, carbohydrate, peptide or amino acid residue); And R when D is nitrogen₄Do not exist.

In another embodiment, 4A, B, C, D, E, F, G and H independently are selected from carbon and nitrogen;

R ₁、R ₂、R ₃、R ₄、R ₈And R₁₀Independently be selected from thiazolinyl, (alkyl of replacement) (alkyl) amino, junior alkyl halides, hydroxyl, alkoxyl, the replacement of heterocyclic radical, the replacement of alkyl, heterocyclic radical, the replacement of hydrogen, halogen, low alkyl group, alkyl, replacement alkoxyl, hydroxyl alkylthio group, nitro, N-alkyl sulfonamide, cyano group ,-COOR₁₁(R wherein₁₁Hydrogen, low alkyl group, aryl, heterocyclic radical, monose or disaccharides) or-CONR₁₂R ₁₃(R wherein₁₂And R₁₃Independently be selected from hydrogen, low alkyl group, aryl, heterocyclic radical, carbohydrate, peptide or amino acid residue).

For compound described here:

Term " low alkyl group " refers to contain side chain or the straight chain acyclic alkyl of 1-10 carbon atom, comprises, for example, methyl, ethyl, propyl group, isopropyl, n-butyl, t-butyl, neopentyl etc.

Term " alkyl " refers to not contain heteroatomic alkyl. Therefore, this term comprises straight chained alkyl, such as methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl etc. This phrase also comprises the branched chain isomer of straight chained alkyl, includes but not limited to following example :-CH (CH₃) ₂、 -CH(CH ₃)(CH ₂CH ₃)、-CH(CH ₂CH ₃) ₂、-C(CH ₃) ₃、-C(CH ₂CH ₃) ₃、-CH ₂CH(CH ₃) ₂、 -CH ₂CH(CH ₃)(CH ₂CH ₃)、-CH ₂CH(CH ₂CH ₃) ₂、-CH ₂C(CH ₃) ₃、-CH ₂C(CH ₂CH ₃) ₃、 -CH(CH ₃)CH(CH ₃)(CH ₂CH ₃)、-CH ₂CH ₂CH(CH ₃) ₂、-CH ₂CH ₂CH(CH ₃)(CH ₂CH ₃)、 -CH ₂CH ₂CH(CH ₂CH ₃) ₂、-CH ₂CH ₂C(CH ₃) ₃、-CH ₂CH ₂C(CH ₂CH ₃) ₃、-CH(CH ₃)CH ₂CH(CH ₃) ₂、 -CH(CH ₃)CH(CH ₃)CH(CH ₃) ₂、-CH(CH ₂CH ₃)CH(CH ₃)CH(CH ₃)(CH ₂CH ₃), etc. This term also comprises cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl, ring octyl group and this class ring of being replaced by above-mentioned straight chain and branched alkyl. This term also comprises multi-ring alkyl, such as, but not limited to, adamantyl, norborny, two ring [2.2.2] octyl groups and this class ring of being replaced by above-mentioned straight chain and branched alkyl. Therefore, the unsubstituted alkyl of phrase comprises primary alkyl, secondary alkyl and tertiary alkyl. Unsubstituted alkyl can be in conjunction with one or more carbon atoms, oxygen atom, nitrogen-atoms and/or the sulphur atom in the parent compound. Preferred unsubstituted alkyl comprises straight chain and branched alkyl and the cycloalkyl that contains 1-20 carbon atom. The unsubstituted alkyl of preferred this class contains 1-10 carbon atom, and preferred this type of group contains 1-5 carbon atom again. Most preferred unsubstituted alkyl comprises straight chain and the branched alkyl that contains 1-3 carbon atom, comprise methyl, ethyl, propyl group and-CH (CH₃) ₂。

Phrase " alkyl of replacement " refers to unsubstituted above defined alkyl, the key that wherein one or more keys that connect carbon or hydrogen are connected non-hydrogen and non-carbon atom substitutes, described non-hydrogen and non-carbon atom such as, but not limited to, halogen atom in the halide is such as F, Cl, Br and I; Phosphorus atoms in the groups such as phosphate and alkyl phosphoric acid dialkyl; Oxygen atom in the groups such as hydroxyl, alkoxyl, aryloxy group and ester group; Sulphur atom in the groups such as mercapto, alkyl sulfur compounds and aromatic yl sulfide, sulfuryl, sulfonyl and sulfoxide group; Nitrogen-atoms in the groups such as amino, amide groups, alkylamine, dialkylamine, arylamine, alkylarylamine, diaryl amine, N-oxide, imidodicarbonic diamide and enamine; Silicon atom in the groups such as trialkylsilkl, di alkylaryl silicyl, alkyl diaryl silicyl and diarye silyl; And other hetero atom in various other groups. The alkyl that replaces comprises that also wherein one or more keys that connect carbon atom or hydrogen atom are connected heteroatomic key and substitute the oxygen in described hetero atom such as carbonyl, carboxyl and the ester group; Nitrogen in the groups such as imines, oxime, hydrazone and nitrile. The preferred alkyl that replaces comprises the alkyl that the key of one or more connection carbon or hydrogen atom is substituted by the key of one or more connection fluorine atoms. An example of the alkyl that replaces is that trifluoromethyl contains the alkyl of trifluoromethyl group with other. The key of those wherein one or more connection carbon of other alkyl or hydrogen atom is connected the alternative group of key of oxygen atom, and therefore, the alkyl of replacement contains hydroxyl, alkoxyl, aryloxy group or heterocyclic oxy group. Other alkyl comprises the alkyl that contains amine, alkylamine, dialkylamine, arylamine, (alkyl) (aryl) amine, diaryl amine, heterocyclic radical amine, (alkyl) (heterocyclic radical) amine, (aryl) (heterocyclic radical) amine or two heterocyclic radical amine groups.

Term " alkoxyl " refers to RO-, and wherein, R for example is alkyl, as mentioned the low alkyl group of definition. The representative example of low-grade alkyl alkoxy comprises methoxyl group, ethyoxyl, t-butoxy etc.

Phrase " alkoxyl of replacement " refers to RO-, and wherein R is the alkyl that is for example replaced by for example halogen. RO is OCF for example₃。

Term " thiazolinyl " refers to contain side chain or the straight chain group that 2-20 carbon atom contains one or more carbon-to-carbon double bonds simultaneously. Representational thiazolinyl comprises prenyl, 2-acrylic (being pi-allyl), 3-methyl-2-butene base, 3,7-dimethyl-2,6-octadienyl, 4,8-dimethyl-3,7-nonadiene base, 3,7,11-trimethyl-2,6,10-dodecane trialkenyl etc.

Phrase " thiazolinyl of replacement " refers to substituted thiazolinyl, for example oneself-5-alkenyl phosphonic acid diethylester (diethyl hex-5-enylphosponate), and other thiazolinyl that is replaced by the alkyl of alkyl or replacement such as dialkyl phosphate or ester such as acetic acid esters.

Phrase " dialkyl amido " refers to by the amino of two alkyl such as the replacement of C1-20 alkyl.

Phrase " dialkyl amido of replacement " refers to by for example dialkyl amido of carboxylate, hydroxyl or alkoxyl replacement.

Term " hydroxyl alkylthio group " refers to be connected with the thio group of hydroxy alkyl, and alkyl wherein is low alkyl group for example. Example have the hydroxyl ethylmercapto group ,-SCH₂CH ₂OH。

Term " N-alkyl sulfonamide " refers to-SO₂The NH alkyl, wherein alkyl is, for example, octyl group.

Side chain or straight chain group that term " alkynyl " refers to contain 2-20 carbon atom and contains one or more carbon-to-carbon triple bonds. Representational alkynyl comprises acetenyl, 2-propynyl (propargyl), 1-propinyl etc.

Term " aryl " refers to not contain heteroatomic aryl. Therefore, this term includes but not limited to following group, such as phenyl, diphenyl, anthryl, naphthyl. Although phrase " unsubstituted aryl " comprises the group that contains condensed ring, naphthalene for example, it does not comprise that one of ring members is connected with the aryl of other group such as alkyl or halo group, for example tolyl is considered to the aryl of replacement hereinafter described here. Preferred unsubstituted aryl is phenyl. Yet unsubstituted aryl can be in conjunction with one or more carbon atoms, oxygen atom, nitrogen-atoms and/or sulphur atom in parent compound.

Phrase " aryl of replacement " has the implication identical with aryl, and the alkyl that replaces is identical with the implication of alkyl. Yet, the aryl that replaces also comprises the aryl that one of them armaticity carbon is combined with an above-mentioned non-carbon or non-hydrogen atom, also comprises the aryl that the armaticity carbon of wherein one or more aryl and defined replacement here and/or unsubstituted alkyl, alkenyl or alkynyl are combined. This comprises following key arrangement, and wherein, two carbon atoms of aryl are combined with two atoms of alkyl, alkenyl or alkynyl and are formed a carbocyclic fused ring system (for example dihydro naphthyl or tetralyl). Therefore, phrase " aryl of replacement " includes, but not limited to tolyl and hydroxyphenyl.

Term " aryl alkyl " refers to be connected with the low alkyl group of aryl. Representational aryl alkyl comprises benzyl, phenethyl, acrinyl, luorobenzyl, fluorobenzene ethyl etc.

Phrase " the aryl aryl of non-condensed " refers to group or substituting group that two aryl mutually do not condense or connect. The example of the aryl aryl compound of non-condensed comprises, for example, benzene biphenyl, diphenyl diazene, 4-methyl mercapto-1-benzene biphenyl, phenoxy group benzene, (2-phenylene-ethynylene) benzene, benzophenone, (4-phenyl fourth-1,3-dialkylene) benzene, phenylbenzyl amine, (phenyl methoxyl group) benzene etc. The aryl aryl of the preferred non-condensed that replaces comprises: 2-(phenyl amino)-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 1; 4-hexichol biphenyl; N-[4-(2-phenylene-ethynylene) phenyl]-the 2-[benzylamino] acetamide; 2-amino-N-[4-(2-phenylene-ethynylene) phenyl] propionamide; 2-amino-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 2-(cyclopropylamino)-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 2-(ethylamino)-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; the 2-[(2-methyl-propyl) amino]-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 5-phenyl-2H-benzo [d] 1; the 3-dioxine (5-phenyl-2H-benzo[d] 1; 3-dioxolene); 2-chloro-1-methoxyl group-4-benzene biphenyl; the 2-[(imidazolyl methyl) amino]-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 4-phenyl-1-phenoxy group benzene; N-(2-aminoethyl) [4-(2-phenylene-ethynylene) phenyl] carboxylic acid amides; the 2-{[(4-fluorophenyl) methyl] amino }-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; the 2-{[(4-aminomethyl phenyl) methyl] amino }-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 4-phenyl-1-(trifluoromethyl) benzene; 1-butyl-4-benzene biphenyl; 2-(cyclohexyl is amino)-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 2-(ethylmethylamino)-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; 2-(butyl is amino)-N-[4-(2-phenylene-ethynylene) phenyl] acetamide; N-[4-(2-phenylene-ethynylene) phenyl]-2-(4-pyridinylamino) acetamide; N-[4-(2-phenylene-ethynylene) phenyl]-2-(quinine-3-base is amino) acetamide; N-[4-(2-phenylene-ethynylene) phenyl] the pyrrolidin-2-yl carboxylic acid amides; 2-amino-3-methyl-N-[4-(2-phenylene-ethynylene) phenyl] butyramide; 4-(4-phenyl fourth-1; the 3-dialkylene) phenyl amine; 2-(dimethylamino)-N-[4-(4-phenyl fourth-1; the 3-dialkylene) phenyl] acetamide; 2-(ethylamino)-N-[4-(4-phenyl fourth-1; the 3-dialkylene) phenyl] acetamide; 4-ethyl-1-benzene biphenyl; 1-[4-(2-phenylene-ethynylene) phenyl] ethyl-1-ketone; N-(1-carbamyl-2-hydroxypropyl) [4-(4-phenyl fourth-1,3-dialkylene) phenyl] carboxylic acid amides; N-[4-(2-phenylene-ethynylene) phenyl] propionamide; 4-methoxybenzene xenyl ketone; phenyl-N-benzamide; (uncle-butoxy)-N-[(4-benzene xenyl) methyl] carboxylic acid amides; 2-(3-benzene biphenylyloxy) ethyl hydroxamic acid; 3-benzene xenyl pyruvate; 1-(4-ethoxyl phenenyl)-4-methoxybenzene and [4-(2-phenylene-ethynylene) phenylpyrrole.

Phrase " the heteroaryl aryl of non-condensed " refers to the aryl aryl of non-condensed, and one of them aryl is heteroaryl. The example of heteroaryl aryl comprises, for example, 2-phenylpyridine, phenylpyrrole, 3-(2-phenylene-ethynylene) pyridine, Phenylpyrazole, 5-(2-phenylene-ethynylene)-1,3-dihydro-pyrimidin-2,4-diketone, 4-phenyl-1,2,3-thiadiazoles, 2-(2-phenylene-ethynylene) pyrazine, 2-phenyl thiophene, phenylimidazole, 3-(2-piperazinyl phenyl) furans, 3-(2,4-dichlorophenyl)-the heteroaryl aryl of the non-condensed that 4-methylpyrrole etc. preferably replaces comprises: 5-(2-phenylene-ethynylene) pyrimidine-2-base amine, 1-methoxyl group-4-(2-thienyl) benzene, 1-methoxyl group-3-(2-thienyl) benzene, 5-methyl-2-phenylpyridine, 5-methyl-3-phenyl-isoxazole azoles, 2-[3-(trifluoromethyl) phenyl] furans, 3-fluoro-5-(2-furyl)-2-methoxyl group-1-third-2-alkenyl benzene, (oxyimino) (5-phenyl (2-thienyl)) methane, 5-[(4-methyl piperazine base) methyl]-2-phenyl thiophene, 2-(4-ethylphenyl) thiophene, 4-methyl mercapto-1-(2-thienyl) benzene, 2-(3-nitrobenzophenone) thiophene, (uncle-butoxy)-N-[(5-phenyl (3-pyridine radicals)) methyl] carboxylic acid amides, hydroxy-n-[(5-phenyl (3-pyridine radicals)) methyl] acid amides, 2-(phenyl methyl mercapto) pyridine and benzyl imidazole.

Phrase " the heteroaryl heteroaryl of non-condensed " refers to the aryl aryl of non-condensed, and wherein two aryl all are heteroaryls. The example of heteroaryl heteroaryl comprises, for example, and 3-Pyridinylimidazoles, 2-imidazole radicals pyrazine etc. The heteroaryl heteroaryl of the preferred non-condensed that replaces comprises: 2-(4-piperazinyl-3-pyridine radicals) furans, diethyl (3-pyrazine-2-base (4-pyridine radicals)) amine and dimethyl 2-[2-(5-methylpyrazine-2-yl) acetenyl] (4-pyridine radicals) } amine.

Phrase " the aryl aryl that condenses " refers to the as mentioned aryl of definition with aryl-condensed and total conjugated. The representational aryl aryl that condenses comprises diphenyl, 4-(1-naphthyl) phenyl, 4-(2-naphthyl) phenyl etc.

Phrase " the heteroaryl aryl that condenses " refers to the as mentioned aryl of definition with heteroaryl-condensed and total conjugated. The representational heteroaryl aryl that condenses comprises quinoline, quinazoline etc.

Phrase " the heteroaryl heteroaryl that condenses " refers to the as mentioned assorted virtue of definition with heteroaryl-condensed and total conjugated. The representational heteroaryl heteroaryl that condenses comprises pyrazolopyrimidine (pyrazalopyrimidine), imidazoquinolie etc.

Term " aryloxy group " refers to RO-, and wherein, R is aryl. Representational alkoxy aryl comprises benzyloxy, phenyl ethoxy etc.

Term " alkoxy aryl " refers to be connected with the lower alkoxy of aryl. Representational alkoxy aryl comprises benzyloxy, phenyl ethoxy etc.

Term " aryloxy group aryl " refers to be connected with the aryl of aryloxy group. Representational aryloxy group aryl comprises 4-Phenoxyphenyl, 3-Phenoxyphenyl, 4-phenoxy group-1-naphthyl, 3-phenoxy group-1-naphthyl etc.

Term " aryloxy group aryl alkyl " refers to be connected with the aryl alkyl of aryloxy group. Representational aryloxy group aryl alkyl comprises 4-Phenoxyphenyl methyl, 3-Phenoxyphenyl methyl, 4-Phenoxyphenyl ethyl, 3-phenoxy group-phenylethyl etc.

Term " alkoxy aryl aryl " refers to be connected with the aryl of alkoxy aryl. Representational alkoxy aryl aryl comprises 4-benzyloxy phenyl, 3-benzyloxy phenyl etc.

Term " alkoxy aryl aryl alkyl " refers to be connected with the aryl alkyl of alkoxy aryl. Representational alkoxy aryl aryl alkyl comprises 4-benzyloxy benzyl, 3-benzyloxy benzyl etc.

Term " cycloalkyl " refers to contain the alcyl of 3-7 carbon atom, includes, but not limited to cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl etc.

Term " cycloalkyl-alkyl " refers to be connected with the low alkyl group of cycloalkyl. The representative example of cycloalkyl-alkyl comprises cyclopropyl methyl, cyclohexyl methyl, 2-(cyclopropyl) ethyl etc.

Term " halogen " refers to iodine, bromine, chlorine or fluorine; " halo " refers to iodo, bromo, chloro or fluoro.

Term " haloalkyl " refers to the as mentioned low alkyl group of definition with at least one halogenic substituent, for example, and chloromethyl, fluoro ethyl or trifluoromethyl etc.

Term " heterocyclic radical " (or heterocycle, heterocycle) expression armaticity and nonaro-maticity cyclic compound, the cyclic compound that comprises monocycle, two rings and many rings, for example, but be not limited to, quininuclidinyl, contain 3 or more annular atomses, wherein one or more are hetero atoms, such as but not limited to N, O and S. Although phrase " unsubstituted heterocyclic radical " comprises annelated heterocycles, such as benzimidazolyl, it does not comprise that one of annular atoms is connected with the heterocyclic radical of other group such as alkyl or halo group, such as the heterocyclic radical of 2-tolimidazole base or replacement. The example of heterocyclic radical comprises, but be not limited to: the first ring of unsaturated 3-8 that contains 1-4 nitrogen-atoms, such as, but not limited to, pyrrole radicals, pyrrolinyl, imidazole radicals, pyrazolyl, pyridine radicals, dihydropyridine base, pyrimidine radicals, pyrazinyl, pyridazinyl, triazolyl (such as 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl etc.), tetrazole radical (such as 1H-TETRAZOLE base, 2H-tetrazole radical etc.); The saturated 3-8 unit ring that contains 1-4 nitrogen-atoms is such as, but not limited to, pyrrolidinyl, imidazolinyl, piperidyl, piperazinyl; Contain the undersaturated heterocycle of condensing of 1-4 nitrogen-atoms, such as, but not limited to, indyl, isoindolyl, indolinyl, indolizine base, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, BTA base; The unsaturated 3-8 unit ring that contains 1-2 oxygen atom and 1-3 nitrogen-atoms, Li as, but be not limited to ， oxazolyl, isoxazolyl, oxadiazolyl (for example 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-Evil di azoly etc.); The first ring of saturated 3-8 that contains 1-2 oxygen atom and 1-3 nitrogen-atoms is such as, but not limited to, morpholinyl; The unsaturated annelated heterocycles that contains 1-2 oxygen atom and 1-3 nitrogen-atoms, for example, benzoxazolyl, Ben Bing oxadiazolyl, benzoxazinyl (for example 2H-1,4-benzoxazinyl etc.); The unsaturated 3-8 unit ring that contains 1-3 sulphur atom and 1-3 nitrogen-atoms is such as, but not limited to, thiazolyl, isothiazolyl, thiadiazolyl group (for example 1,2,3-thiadiazolyl group, 1,2,4-thiadiazolyl group, 1,3,4-thiadiazolyl group, 1,2,5-thiadiazolyl group etc.); The first ring of saturated 3-8 that contains 1-2 sulphur atom and 1-3 nitrogen-atoms is such as, but not limited to, thiazolidinyl; The first ring of saturated and unsaturated 3-8 that contains 1-2 sulphur atom, for example, but be not limited to thienyl, dihydro dithia cyclohexadienyl (dihydrodithiinyl), dihydro two thio groups (dihydrodithionyl), thiophane, tetrahydric thiapyran; The unsaturated annelated heterocycles that contains 1-2 sulphur atom and 1-3 nitrogen-atoms, such as, but not limited to, benzothiazolyl, diazosulfide base, benzothiazine base (2H-1 for example, 4-benzothiazine base etc.), dihydrobenzo thiazinyl (for example 2H-3,4-dihydrobenzo thiazinyl etc.); The first ring of unsaturated 3-8 that contains oxygen atom is such as, but not limited to, furyl; The unsaturated annelated heterocycles that contains 1-2 oxygen atom, for example benzo dioxo base (benzodioxolyl) (for example 1,3-benzo dioxo base etc.); The first ring of unsaturated 3-8 that contains an oxygen atom and 1-2 sulphur atom is such as, but not limited to, dihydro oxathiin base; The first ring of saturated 3-8 that contains 1-2 oxygen atom and 1-2 sulphur atom is such as Isosorbide-5-Nitrae-thioxane; The unsaturated condensed ring that contains 1-2 sulphur atom is such as benzothienyl, benzo dithia cyclohexadienyl; And the unsaturated annelated heterocycles that contains an oxygen atom and 1-2 oxygen atom, such as benzo oxathiin base. Heterocyclic radical also comprises above-mentioned group, and wherein, the two bonds of the one or more S atoms in the ring and one or two oxygen atom are closed (sulfoxide and sulfone). For example, heterocyclic radical comprises thiophane, thiophane oxide and thiophane 1,1-dioxide. Preferred heterocyclic radical contains 5 or 6 annular atomses. Preferred heterocyclic radical comprises morpholine, piperazine, piperidines, pyrrolidines, imidazoles, pyrazoles, 1,2,3-triazole, 1,2,4-triazole, tetrazolium, thiomorpholine, thiomorpholine (wherein, the sulphur atom of thiomorpholine is combined with one or more oxygen atoms), pyrroles, homopiperazine, oxazolidine-2-ketone, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furans and oxolane.

Phrase " heterocyclic radical of replacement " refers to heterocyclic radical defined above, one of them ring members with above be combined at the non-hydrogen atom described in the aryl of the alkyl that replaces and replacement. Its example includes, but not limited to 2-tolimidazole base, 5-tolimidazole base, 5-chloro benzothiazole base, 1-methyl piperazine base and 2-chloropyridine base. " amino-sulfonyl " refers to group-S (O)₂-NH ₂ " amino-sulfonyl that replaces " refers to group-S (O)₂-NRR ', wherein R is low alkyl group, R ' is hydrogen or low alkyl group. Term " aryl alkyl amino sulfonyl aryl " refers to group-aryl-S (O)₂-NH-aralkyl, wherein said aralkyl are rudimentary aralkyl.

" carbonyl " refer to divalent group-C (O)-.

" carbonyl oxygen base " is often referred to group-C (O)-O-. This group comprises ester-C (O)-O-R, and wherein R is low alkyl group, cycloalkyl, aryl or rudimentary aralkyl. Term " carbonyl oxygen basic ring alkyl " is often referred to " carbonyl oxygen base carbocyclic ring alkyl " and " carbonyl oxygen base Heterocyclylalkyl ", that is, wherein R is respectively carbocyclic ring alkyl or Heterocyclylalkyl. Term " aryl-carbonyl oxygen " refers to group-C (O)-O-aryl, and wherein aryl is isocyclic aryl or the heterocyclic aryl of monocycle or many rings. Term " aralkyl carbonyl oxygen base " refers to group-C (O)-O-aralkyl, and wherein aralkyl is rudimentary aralkyl.

Term " sulfonyl " refers to group-SO₂-. " alkyl sulphonyl " refers to have structure-SO₂The sulfonyl of the replacement of R-, wherein R is alkyl. The alkyl sulphonyl that is used for the compounds of this invention normally main chain contains the low alkyl group sulfonyl of 1-6 carbon atom. Therefore, the alkyl sulphonyl that is used for the compounds of this invention generally includes, for example, and methyl sulphonyl (being that R is methyl), ethylsulfonyl (being that R is ethyl), sulfonyl propyl base (being that R is propyl group) etc. Term " aryl sulfonyl " refers to group-SO₂-aryl. Term " aralkyl sulfonyl " refers to group-SO₂-aralkyl, wherein aralkyl is rudimentary aralkyl. Term " sulfonamido " refers to-SO₂NH ₂。

Term " carbonylamino " refer to divalent group-NH-C (O)-, wherein, the hydrogen atom of amide nitrogen can be replaced by low alkyl group, aryl or rudimentary aralkyl in the carbonylamino. This group comprises that (NH-C (O)-O-R) and acid amides-NH-C (O)-O-R two parts, wherein R is straight or branched low alkyl group, cycloalkyl or aryl or rudimentary aralkyl to carbamate. Term " lower alkylcarbonyl is amino " refers to alkyl-carbonyl-amino, and wherein R is the low alkyl group that contains 1-6 carbon atom in the backbone structure. Term " aryl-amino-carbonyl " refers to group-NH-C (O)-R, and wherein R is aryl. Similarly, term " aromatic alkyl carbonyl is amino " refers to carbonylamino, and wherein R is rudimentary aralkyl.

Term " guanidine radicals " refer to derived from guanidine H2N-C (=NH)-NH₂Part. This part comprise be incorporated into the part on the nitrogen-atoms of formal two keys (" 2 " of guanidine-position, for example, diamino methylene amino (H2N)₂C=NH-) and be incorporated into part on one of two nitrogen-atoms with formal singly-bound (" 1-" of guanidine and/or " 3 "-position, for example, H₂N-C (=NH)-NH-). Hydrogen atom on one of nitrogen-atoms can by suitable substituting group, replace such as low alkyl group, aryl or rudimentary aralkyl.

Representational cyclo-imino and heterocycle imino group comprise, for example, and those shown in the below. These cyclo-iminos and heterocycle imino group also can be substituted and can be combined in diverse location, and in conjunction with the description here, this is apparent for the technical staff who is proficient in organic chemistry and pharmaceutical chemistry field.

With

The amidino groups of representational replacement and heterocycle amidino groups comprise, for example, and those shown in the below. These amidino groups and heterocycle amidino groups also can be substituted, and in conjunction with the description here, this is apparent for the technical staff who is proficient in organic chemistry and pharmaceutical chemistry field.

With

The alkyl-carbonyl-amino of representational replacement, alkoxycarbonyl amino, aminoalkoxide carbonylamino and aryl-amino-carbonyl comprise, for example, and those shown in the below. These groups also can be substituted, and in conjunction with the description here, this is apparent for the technical staff who is proficient in organic chemistry and pharmaceutical chemistry field.

With

The amino carbonyl of representational replacement comprises, for example, and those shown in the below. These heterocyclic radicals also can be substituted, and in conjunction with the description here, this is apparent for the technical staff who is proficient in organic chemistry and pharmaceutical chemistry field.

With

The alkoxy carbonyl of representational replacement comprises, for example, and those shown in the below. These alkoxy carbonyls also can be substituted, and in conjunction with the description here, this is apparent for the technical staff who is proficient in organic chemistry and pharmaceutical chemistry field.

With

" replacement " refers to clearly replace hydrogen with one or more unit prices or divalent group. Suitable substituting group comprises this paper to described those groups of concrete group, and alkyl of hydroxyl, nitro, amino, imino group, cyano group, halo, sulfo-, sulfo-amino, amidino groups, imino group, oxo, oxalyl amido, methoxalyl amido, imino group, guanidine radicals, sulfonamido, carboxyl, formoxyl, alkyl, replacement, junior alkyl halides, lower alkoxy, halogenated lower alkoxy, lower alkoxy-alkyl, alkyl-carbonyl, aryl carbonyl, aromatic alkyl carbonyl, heteroaryl carbonyl, heteroarylalkyl carbonyl, alkylthio group, aminoalkyl, cyano group alkyl, benzyl, pyridine radicals, pyrazolyl, pyrroles, thiophene, imidazole radicals etc.

Term " coupling part " refers to covalent bond or non-cyclisation divalent group, for example ,-CO-,-O-,-S-,-CH₂-,-NH-and replacing as herein defined or unsubstituted alkyl, thiazolinyl, alkynyl, carbonyl, alkoxy carbonyl.

Term " SMIP compound " refers to that little molecular immune strengthens compound, comprises that molecular weight is lower than about MW 1000 g/mol, the micromolecular compound that can stimulate or regulate the short inflammatory response of patient of preferred MW 800g/mol. In one embodiment, described SMIP compound can be made cell factor by the stimulation human peripheral blood monocyte. Preferred SMIP compound and derivative thereof comprise; for example, (ABIQ) compound, hydroxyl phthalimide (hydraphthalimide) compound, pyrazoles pyrimidine compound, Quinazolinone compounds, quinoxaline compound, triaizine compounds, tetrahydro pyrrolidine and quinoxaline compound, azole compounds, biphenyl ketonic compound, phytosterin compound and isoxazole compound of amino azepine vinyl compound, benzazole compound, acyl piperazine compound, indole dione compound, tetrahydroisoquinoline (THIQ) compound, anthraquinone compounds, indandione compound, phthalimide (pthalimide) compound, benzo ring dione compounds, aminobenzimidazole quinolinone (aminobenzimidazole quinolinone).

Term " SMIS compound " refers to little molecular immune inhibitor compound, comprises that molecular weight is lower than about MW 1000 g/mol, the micromolecular compound that can suppress or regulate the short inflammatory response of patient of preferred MW 800g/mol.

Acyl piperazine compound described in the application comprises the compound of formula (III), and is as described below:

Wherein,

R ₉Be selected from and replace or unsubstituted aryl, heteroaryl, aryl alkyl, aryl alkenyl, heteroaryl alkyl and heteroaryl thiazolinyl;

R ₁₀To replace or unsubstituted alkyl;

N is the integer of 0-2; With

If D₁Carbon, D then₂Oxygen, D₃Do not exist, and D₄The heteroaryl aryl that is selected from replacement or unsubstituted aryl, heteroaryl, carbocylic radical, alkoxy aryl, the aryl aryl that condenses, the aryl heteroaryl that condenses and condenses; Or,

If D₁Nitrogen, D then₂Nitrogen, D₄Do not exist, and D₃Be selected from replacement or unsubstituted aryl, heteroaryl, carbocylic radical, alkoxy aryl, the aryl aryl that condenses, the aryl heteroaryl that condenses and the heteroaryl aryl that condenses.

Indole dione compound described in the application comprises the compound of formula (IV), and is as described below:

Wherein,

R ₁₁And R₁₂Independently be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid (carboxcyclic acid) and replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical; With

R ₁₃Be selected from and replace or unsubstituted aryl, heteroaryl, aryl alkyl, heteroaryl alkyl, heterocyclic radical, heterocyclic radical alkyl and alkyl benzyl.

Tetrahydroisoquinoline described in the application (THIQ) compound comprises the compound of formula (V), and is as described below:

Wherein,

L is covalent bond or is selected from-CH₂-。-CO-、-O-、-S-、CHF、-NH-、-NR ₂₀-, R wherein₂₀It is low alkyl group;

R ₁₄Be selected from hydrogen, halogen or replacement or unsubstituted alkyl;

R ₁₅Be selected from replacement or unsubstituted carbocylic radical, aryl, aryl alkyl, alkoxy aryl, heteroaryl, heterocyclic radical;

R ₁₆Be selected from hydrogen, halogen or replacement or unsubstituted alkyl;

R ₁₇Be selected from hydrogen, halogen or replacement or unsubstituted alkyl; With

R ₁₈And R₁₉Independently be selected from_H, hydroxyl, halogen, alkoxyl, amino, unsubstituted alkyl, replacement alkyl and alkyl amino.

Benzo ring dione compounds described in the application comprises the compound of formula (VI), and is as described below:

Wherein,

E is selected from NR₂₅Or CR₂₆R ₂₇；

R ₂₁、R ₂₃And R₂₄Independently be selected from alkyl and the alkyl amino of H, hydroxyl, halogen, alkoxyl, amino, unsubstituted alkyl, replacement;

R ₂₂The alkyl and alkyl amino, aryl alkyl, heteroaryl alkyl, aryl, heteroaryl, aryl carbonyl, heterocyclic radical, heterocyclic radical alkyl and the heteroaryl carbonyl that are selected from H, hydroxyl, halogen, alkoxyl, amino and do not replace or replace;

R ₂₅Be selected from and replace or unsubstituted aryl, heteroaryl, heterocyclic radical, carbocylic radical, aryl alkyl, heteroaryl alkyl and Heterocyclylalkyl;

R ₂₆Be selected from H, halogen, hydroxyl, amino and replacement or unsubstituted alkyl, carbonylic alkyl and alkyl-carbonyl alkyl; With

R ₂₇Be selected from aryl, aryl alkyl, heteroaryl alkyl, heterocyclic radical, heterocyclic radical alkyl, carbocylic radical, aryl alkyl carbonyl and aromatic yl alkyl carbonyl.

Amino azepine vinyl compound described in the application comprises the compound of formula (VII), and is as described below:

Wherein,

G is S or NH;

R ₂₈Be selected from H and replacement or unsubstituted alkyl, aryl, heteroaryl, heteroaryl alkyl, aryl alkyl, carbocylic radical, carbocylic radical alkyl, heterocyclic radical and heterocyclic radical alkyl;

Q be selected from heteroaryl, heterocyclic radical, the replacement of aryl, heteroaryl, the replacement of the alkyl of hydrogen, replacement, unsubstituted alkyl and aryl, replacement heterocyclic radical, condense or the heteroaryl heteroaryl of aryl heteroaryl, heteroaryl heteroaryl and the replacement of the aryl aryl of the aryl aryl of non-condensed, replacement, aryl heteroaryl, replacement;

V ₁Be selected from alkyl, the alkyl that replaces, aryl, the aryl that replaces, aryl alkyl, the aryl alkyl that replaces, heteroaryl, the heteroaryl that replaces, heteroaryl alkyl, the heteroaryl alkyl that replaces, alkoxyl, the alkoxyl that replaces, amino carbonyl, alkoxy carbonyl, the carboxyl sulfonyl, mesyl and replacement or unsubstituted alkyl-carbonyl, aryl carbonyl, aromatic alkyl carbonyl, the heteroaryl carbonyl, the heteroarylalkyl carbonyl, alkyl carbonyl oxy, aryl-carbonyl oxygen, aralkyl carbonyl oxygen base, heteroaryl carbonyl oxygen base, heteroarylalkyl carbonyl oxygen base, alkyl amino carbonyl oxy, arylamino carbonyl oxygen base, formoxyl, lower alkylcarbonyl, elementary alkoxy carbonyl, amino carbonyl, aminoaryl, alkyl sulphonyl, sulfonamido, aminoalkoxy, alkyl amino, heteroaryl amino, alkyl-carbonyl-amino, alkyl amino-carbonyl is amino, aromatic yl aminocarbonyl is amino, aromatic alkyl carbonyl is amino, the heteroaryl carbonylamino, aryl-amino-carbonyl, the ring amidino groups, cycloalkyl, cyclo-imino, aryl sulfonyl or aromatic yl sodium sulfonamido; With

V ₂Be selected from hydrogen; halogen; alkyl; the alkyl that replaces; aryl; the aryl that replaces; aryl alkyl; the aryl alkyl that replaces; heteroaryl; the heteroaryl that replaces; heteroaryl alkyl; the heteroaryl alkyl that replaces; alkoxyl; the alkoxyl that replaces; amino carbonyl; alkoxy carbonyl; the carboxyl sulfonyl; mesyl and replacement or unsubstituted alkyl-carbonyl; aryl carbonyl; aromatic alkyl carbonyl; the heteroaryl carbonyl; the heteroarylalkyl carbonyl; alkyl carbonyl oxy; aryl-carbonyl oxygen; aralkyl carbonyl oxygen base; heteroaryl carbonyl oxygen base; heteroarylalkyl carbonyl oxygen base; alkyl amino carbonyl oxy; arylamino carbonyl oxygen base; formoxyl; lower alkylcarbonyl; elementary alkoxy carbonyl; amino carbonyl; aminoaryl; alkyl sulphonyl; sulfonamido; aminoalkoxy; alkyl amino; heteroaryl amino; alkyl-carbonyl-amino; alkyl amino-carbonyl is amino; aromatic yl aminocarbonyl is amino; aromatic alkyl carbonyl is amino; the heteroaryl carbonylamino; aryl-amino-carbonyl; the ring amidino groups; cycloalkyl; cyclo-imino; aryl sulfonyl and aromatic yl sodium sulfonamido.

Lactam compound described in the application comprises the compound of formula (VIII), and is as described below:

Wherein,

W ₁Be selected from-OH ,-OR₃₆、-NR ₃₇R ₃₈；

W ₂Be selected from O, S, NR₃₉

R ₂₉And R₃₀Form together one and replace or unsubstituted 5-6 unit ring, this ring all is carbon atom or contains at least one O, N or the S atom;

R ₃₅And R₃₉Can be identical or different, and be selected from H ,-OH, replacement and unsubstituted alkyl, replacement and unsubstituted aryl ,-C (=O) H ,-C (=O)-alkyl or-C (=O)-aryl;

R ₃₁、R ₃₂、R ₃₃And R₃₄Can be identical or different, and independently be selected from H, Cl, Br, F, I ,-NO₂、-CN、 -OH、-OR ₄₀、-NR ₄₁R ₄₂、-C(＝O)R ₄₃-SH, replace and unsubstituted amidino groups, replace and unsubstituted guanidine radicals, replace and unsubstituted alkyl, replace and unsubstituted aryl, replace and unsubstituted thiazolinyl, replace and unsubstituted alkynyl, replace and unsubstituted heterocyclic radical, replace and unsubstituted alkyl amino alkyl, replace and unsubstituted dialkyl aminoalkyl, replace and unsubstituted arylamino alkyl, replace and unsubstituted ammonia diaryl base alkyl, replace and unsubstituted (alkyl) (aryl) aminoalkyl, replace and unsubstituted heterocyclic radical alkyl, replace and unsubstituted aminoalkyl, replacement and unsubstituted Heterocyclylaminoalksubstituted, replace and unsubstituted two Heterocyclylaminoalksubstituted, replace and unsubstituted (alkyl) (heterocyclic radical) aminoalkyl, replace and unsubstituted (aryl) (heterocyclic radical) aminoalkyl, replace and unsubstituted hydroxy alkyl, replace and unsubstituted alkoxyalkyl, replace and unsubstituted aryloxy alkyl and replacement or unsubstituted heterocyclic oxy group alkyl;

R ₃₆Be selected from replace and unsubstituted alkyl, replacement and unsubstituted aryl, replacement and unsubstituted heterocyclic radical, replacement and unsubstituted heterocyclic radical alkyl ,-C (=O) H ,-C (=O)-alkyl ,-C (=O)-aryl ,-C (=O) the O-alkyl ,-C (=O) the O-aryl ,-C (=O) NH₂,-C (=O) NH (alkyl) ,-C (=O) NH (aryl) ,-C (=O) N (alkyl)₂,-C (=O) N (aryl)₂,-C (=O) N (alkyl) (aryl) ,-NH₂,-NH (alkyl) ,-NH (aryl) ,-N (alkyl)₂,-N (alkyl) (aryl) ,-N (aryl)₂,-C (=O) NH (heterocyclic radical) ,-C (=O) N (heterocyclic radical)₂,-C (=O) N (alkyl) (heterocyclic radical) or-C (=O) N (aryl) (heterocyclic radical);

R ₃₇Be selected from H, replacement and unsubstituted alkyl, replacement and unsubstituted aryl or replacement and unsubstituted heterocyclic radical;

R ₃₈Be selected from H, replacement and unsubstituted alkyl, replacement and unsubstituted aryl, replacement and unsubstituted heterocyclic radical ,-OH, alkoxyl, aryloxy group ,-NH₂, replacement and unsubstituted heterocyclic radical alkyl, replacement and unsubstituted aminoalkyl, replacement and unsubstituted alkyl amino alkyl, replacement and unsubstituted dialkyl aminoalkyl, replacement and unsubstituted arylamino alkyl, replacement and unsubstituted ammonia diaryl base alkyl, replacement and unsubstituted (alkyl) (aryl) aminoalkyl, replacement and unsubstituted alkyl amino, replacement and unsubstituted arylamino, replacement and unsubstituted dialkyl amido, replacement and unsubstituted ammonia diaryl base, replacement and unsubstituted (alkyl) (aryl) be amino ,-C (=O) H ,-C (=O)-alkyl ,-C (=O)-aryl ,-C (=O) the O-alkyl ,-C (=O) the O-aryl ,-C (=O) NH₂,-C (=O) NH (alkyl) ,-C (=O) NH (aryl) ,-C (=O) N (alkyl)₂,-C (=O) N (aryl)₂,-C (=O) N (alkyl) (aryl) ,-C (=O)-heterocyclic radical ,-C (=O)-the O-heterocyclic radical ,-C (=O) NH (heterocyclic radical) ,-C (=O)-N (heterocyclic radical)₂,-C (=O)-N (alkyl) (heterocyclic radical) ,-C (=O)-N (aryl) (heterocyclic radical, replacement and unsubstituted Heterocyclylaminoalksubstituted, replacement and unsubstituted two Heterocyclylaminoalksubstituted, replacement and unsubstituted (alkyl) (heterocyclic radical) aminoalkyl, replacement and unsubstituted (aryl) (heterocyclic radical) aminoalkyl, replacement and unsubstituted hydroxy alkyl, replacement and unsubstituted alkoxyalkyl, replacement and unsubstituted aryloxy alkyl or replacement and unsubstituted heterocyclic oxy group alkyl;

R ₄₁Be selected from H, replacement and unsubstituted alkyl, replacement and unsubstituted aryl or replacement and unsubstituted heterocyclic radical;

R ₄₂Be selected from H, replacement and unsubstituted alkyl, replacement and unsubstituted aryl, replacement and unsubstituted heterocyclic radical ,-C (=O) H ,-C (=O)-alkyl ,-C (=O)-aryl ,-C (=O) NH₂,-C (=O) NH (alkyl) ,-C (=O) NH (aryl) ,-C (=O) N (alkyl)₂,-C (=O) N (aryl)₂,-C (=O) N (alkyl) (aryl) ,-C (=O) the O-alkyl ,-C (=O) O-aryl, replacement and unsubstituted aminoalkyl, replacement and unsubstituted alkyl amino alkyl, replacement and unsubstituted dialkyl aminoalkyl, replacement and unsubstituted arylamino alkyl, replacement and unsubstituted ammonia diaryl base alkyl, replacement and unsubstituted (alkyl) (aryl) aminoalkyl, replacement and unsubstituted heterocyclic radical alkyl ,-C (=O)-heterocyclic radical ,-C (=O)-the O-heterocyclic radical ,-C (=O) NH (heterocyclic radical) ,-C (=O)-N (heterocyclic radical)₂,-C (=O)-N (alkyl) (heterocyclic radical) ,-C (=O)-N (aryl) (heterocyclic radical), replacement and unsubstituted Heterocyclylaminoalksubstituted, replacement and unsubstituted two Heterocyclylaminoalksubstituted, replacement and unsubstituted (heterocyclic radical) (alkyl) aminoalkyl, replacement and unsubstituted (heterocyclic radical) (aryl) aminoalkyl, replacement and unsubstituted hydroxy alkyl, replacement and unsubstituted alkoxyalkyl, replacement and unsubstituted aryloxy alkyl or replacement and unsubstituted heterocyclic oxy group alkyl; With

R ₄₃Be selected from H ,-NH₂,-NH (alkyl) ,-NH (aryl) ,-N (alkyl)₂,-N (aryl)₂,-N (alkyl) (aryl) ,-NH (heterocyclic radical) ,-N (heterocyclic radical) (alkyl) ,-N (heterocyclic radical) (aryl) ,-N (heterocyclic radical)₂, replacement and unsubstituted alkyl, replacement and unsubstituted aryl ,-OH, replace and unsubstituted alkoxyl, replacement and unsubstituted heterocyclic radical, replacement and unsubstituted aryloxy group, heterocyclic oxy group ,-NHOH ,-N (alkyl) OH ,-N (aryl) OH ,-N (alkyl) O-alkyl ,-N (aryl) O-alkyl ,-N (alkyl) O-aryl or-N (aryl) O-aryl.

Preferred R₂₉And R₃₀Forming together one replaces or unsubstituted phenyl ring.

Hydroxyl phthalamide (Hydropthalamide) compound described in the application comprises the compound of formula (IX), and is as described below:

Wherein,

R ₄₄Be selected from the aryl heteroaryl of replacement or unsubstituted aryl, heteroaryl, aryl alkyl, heteroaryl alkyl, the aryl aryl that condenses, the aryl aryl of non-condensed, the heteroaryl aryl that condenses, the heteroaryl aryl of non-condensed, the aryl heteroaryl that condenses or non-condensed;

R ₄₅、R ₄₇、R ₄₉And R₅₁Can be identical or different, and independently be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid and replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl or carbocylic radical; With

R ₄₆、R ₄₈、R ₅₀And R₅₂Can be identical or different, and independently be selected from H, halogen and replacement or unsubstituted alkyl.

Biphenyl ketonic compound described in the application comprises the compound of formula (X), and is as described below:

Wherein,

R ₅₃Independently be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid or replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl, carbocylic radical;

R ₅₄Independently be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid or replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl, carbocylic radical; With

O and p are the integers of 0-4.

The De isoxazole compound comprises the compound of formula (XI) described in the application, and is as described below:

Wherein,

R ₅₅Be selected from and replace or unsubstituted aryl, aryl alkyl, heteroaryl, heteroaryl alkyl, heterocyclic radical and heterocyclic radical alkyl;

R ₅₆Be selected from and replace or unsubstituted aryl, aryl alkyl, heteroaryl, heteroaryl alkyl, heterocyclic radical and heterocyclic radical alkyl; With

R ₅₇Be selected from H, halogen, hydroxyl or replacement or unsubstituted alkyl, aryl, heteroaryl, heterocyclic radical and carbonyl.

Sterid described in the application comprises the compound of formula (XII), and is as described below:

Wherein,

R ₅₈Be selected from and replace or unsubstituted aryl, aryl alkyl, heteroaryl, heteroaryl alkyl, heterocyclic radical and heterocyclic radical alkyl.

Preferred R₅₈It is the pyrones substituting group.

Quinazolinone compounds described in the application comprises the compound of formula (XIII), and is as described below:

Wherein,

R ₅₉Be selected from H, halogen, hydroxyl and replacement or unsubstituted alkyl, aminoalkyl, alkyl amino alkyl, alkoxyl, dialkyl aminoalkyl, hydroxy alkyl, thiazolinyl, alkynyl, carbocylic radical, carbocylic radical alkyl, heterocyclic radical and heterocyclic radical alkyl;

R ₆₀Be selected from and replace or unsubstituted aryl, heteroaryl, aryl alkyl, heteroaryl alkyl and heterocyclic radical alkyl; With

R ₆₁、R ₆₂、R ₆₃And R₆₄Can be identical or different, and independently be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid or replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical.

Azole compounds described in the application comprises the compound of formula (XIV), and is as described below:

Wherein,

R ₆₅Be selected from H, hydroxyl and replacement or unsubstituted alkyl, aryl, heteroaryl, heteroaryl alkyl, aryl alkyl, heteroaryl amino alkyl, arylamino alkyl, heteroaryloxy alkyl and aryloxy alkyl;

R ₆₆、R ₆₇、R ₆₈And R₆₉Can be identical or different, and independently be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid or replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical.

Other preferred azole compounds comprises the compound shown in the formula (XV):

Wherein:

K ₁Nitrogen, oxygen or the optional carbon that replaces;

W do not exist or be selected from-O-,-S-,-S (O)-,-SO₂-、-NH-、-NH-CO-、-NR’CO-、-NHSO ₂-、 -NR’SO ₂-、-CO-、-CO ₂-、-CH ₂-、-CF ₂-, CHF ,-CONH-,-CONR '-or-NR '-, wherein R ' is alkyl, cycloalkyl, aryl, heteroaryl, the heterocycle of alkyl, replacement;

Ar is optional aryl, heteroaryl or the protecting group that replaces;

R ₇₀And R₇₀' independently be selected from hydrogen and methyl;

R ₇₁、R ₇₂、R ₇₃And R₇₄Independently be selected from hydrogen, hydroxyl and the optional low alkyl group that replaces, ring low alkyl group, ring aminoalkyl, alkyl amino alkyl, lower alkoxy, amino, alkyl amino, alkyl-carbonyl, aryl carbonyl, aromatic alkyl carbonyl, heteroaryl carbonyl, heteroarylalkyl carbonyl, aryl or heteroaryl;

R ₇₅And R₇₈Independently be selected from hydrogen, halo and optional low alkyl group, cycloalkyl, alkoxyl, amino, aminoalkoxy, carbonyl oxygen base, amino carbonyl oxygen base, alkyl-carbonyl-amino, aryl-amino-carbonyl, aromatic alkyl carbonyl amino, heteroaryl carbonylamino, heteroarylalkyl carbonylamino, cyclo-imino, heterocycle imino group, amidino groups, ring amidino groups, heterocycle amidino groups, the guanidine radicals that replaces, aryl, heteroaryl, Heterocyclylalkyl, heterocycle carbonyl oxygen base, heteroaryl carbonyl oxygen base and aromatic yl sodium sulfonamido;

R ₇₆Be selected from the heterocyclic radical of heteroaryl, heterocyclic radical and the replacement of hydrogen, aryl, heteroaryl, replacement;

R ₇₇Be selected from hydrogen, hydroxyl, halo, carboxyl, nitro, amino, acylamino-, amidino groups, imino group, cyano group, sulfonyl, mesyl, or replacement or unsubstituted alkyl, alkoxyl, alkyl-carbonyl, aryl carbonyl, aromatic alkyl carbonyl, the heteroaryl carbonyl, the heteroarylalkyl carbonyl, alkyl carbonyl oxy, aryl-carbonyl oxygen, aralkyl carbonyl oxygen base, heteroaryl carbonyl oxygen base, heteroarylalkyl carbonyl oxygen base, alkyl amino carbonyl oxy, arylamino carbonyl oxygen base, formoxyl, lower alkylcarbonyl, elementary alkoxy carbonyl, amino carbonyl, aminoaryl, alkyl sulphonyl, sulfonamido, aminoalkoxy, alkyl amino, heteroaryl amino, alkyl-carbonyl-amino, alkyl amino-carbonyl is amino, aromatic yl aminocarbonyl is amino, aromatic alkyl carbonyl is amino, the heteroaryl carbonylamino, aryl-amino-carbonyl, heteroaryl carbonylamino cyclic amide base, epithio is for amido, the ring amidino groups, the heterocycle amidino groups, cycloalkyl, cyclo-imino, the heterocycle imino group, guanidine radicals, aryl, heteroaryl, heterocycle, Heterocyclylalkyl, aryl sulfonyl and aromatic yl sodium sulfonamido;

Anthraquinone compounds of the present invention comprises, for example, and the compound of formula (XVI):

Wherein,

R ₇₉、R ₈₀、R ₈₁And R₈₂Can be identical or different, and independently being selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid, and replaces or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, sulfonyl, amino-sulfonyl, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical; With

R ₈₃And R₈₄Form together replacement or unsubstituted 5-6 unit ring, this ring all is carbon atom or contains 1-2 hetero atom that is selected from O, S or N.

The described quinoxaline compound of the application comprises the unconjugated tricyclic compound of part, and can choose wantonly by the preferred embodiment of nitrogen heteroatom replacement ， quinoxaline shown in (XVII):

Wherein,

J ₁C or N,

J ₁' be selected from heteroaryl and the unsubstituted heteroaryl of the aryl of H, replacement, unsubstituted aryl, replacement;

J ₂C or N,

J ₂' be selected from heteroaryl and the unsubstituted heteroaryl of the aryl of H, replacement, unsubstituted aryl, replacement;

J ₃Be selected from-CO-,-NH-or-N=;

If J₄Be-O-, then J₄' do not exist; Or,

If J₄Be=C-, then J₄' be selected from H and replacement or unsubstituted alkyl, alkoxyl, aryl, heteroaryl, heteroaryl alkyl, aryl alkyl, aminoalkyl, alkyl amino and alkylthio group; With

R ₈₅、R ₈₆、R ₈₇、R ₈₈And R₈₉Can be identical or different, and independently be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid or replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, sulfonyl, amino-sulfonyl, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical.

Triaizine compounds refers to 6 yuan of heterocycles replacing, and whole ring is replaced by 3 nitrogen-atoms. The preferred embodiments of the invention comprise structural formula (XVIII), (XIX) and (XX) shown those:

Wherein,

R ₉₀Be selected from and replace or unsubstituted alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heteroaryl alkyl, heteroaryl thiazolinyl, aryl alkyl and aryl alkenyl;

R ₉₁And R₉₃Independently be selected from H and unsubstituted alkyl;

R ₉₁It is aryl; Preferred phenyl,

Wherein,

R ₉₄Be selected from H, amino, alkyl, aminoalkyl and halogen;

R ₉₅Be selected from and replace or unsubstituted aryl, arylamino, aryl-alkyl amino, heteroaryl, heteroaryl amino and assorted alkyl amino;

R ₉₆And R₉₇Independently be selected from H, halogen or alkyl, preferable methyl; Or,

R ₉₆Can be directly and nitrogen-atoms form two keys, pointed such as the dotted line in the above-mentioned structural formula; With

Wherein,

R ₉₈Be selected from alkyl or the unsubstituted alkyl of H, replacement; Preferable methyl,

R ₉₉Be selected from alkyl or the unsubstituted alkyl of H, replacement; Preferred ethyl,

R ₁₀₀Be selected from and replace or unsubstituted aryl, heteroaryl, alkoxy aryl, aryl alkyl or heteroaryl alkyl.

The described benzazole compound of the application comprises the described compound of following formula (XXI):

Wherein,

A is selected from-O-,-S-,-NH-or-NR₈-；

W is selected from-CH₂-,-O-,-S-,-NH-or-NR₈-；

R ₇Be selected from the aryl aryl of non-condensed of the aryl heteroaryl that condenses of the carbocylic radical carbocylic radical of carbocylic radical, non-condensed, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, replacement, the unsubstituted aryl heteroaryl that condenses, replacement or the aryl aryl of unsubstituted non-condensed;

R ₆Be selected from and replace or unsubstituted aryl and heteroaryl; With

R ₈Independent of replacing or unsubstituted alkyl.

The described Pyrazolopyrimidine compound of the application comprises the compound shown in the following formula (XXII):

Wherein,

R ₁₀₁Be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid or replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, sulfonyl, amino-sulfonyl, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical;

R ₁₀₂Be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid or replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical;

R ₁₀₃Be selected from H, nitro, halogen, amino, hydroxyl, cyano group, carbocyclic ring acid, trifluoromethyl and replacement or unsubstituted alkyl, aryl, heteroaryl, alkoxyl, alkyl-carbonyl, alkyl-carbonyl-amino, alkyl amino-carbonyl, amino carbonyl, alkoxy aryl, heteroaryl alkoxyl, alkyl amino, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl and carbocylic radical;

R ₁₀₄Be selected from H and replacement or unsubstituted aryl, heteroaryl, alkoxy aryl, heteroaryl alkoxyl, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl, carbocylic radical alkyl, carbocylic radical;

R ₁₀₅Be selected from H or replacement or unsubstituted aryl, heteroaryl, alkoxy aryl, heteroaryl alkoxyl, aryl-alkyl amino, arylamino, heteroaryl amino, heteroaryl amino alkyl, heterocyclic radical, heterocyclic radical alkoxyl, heterocyclic radical alkyl, carbocylic radical alkyl, carbocylic radical;

Wherein, R₁₀₄And R₁₀₅In have at least one not to be H.

Following method 1 and 2 are seen in description by the SMIP compound of method discriminating in external (cell or acellular test method(s)) or the body.

The pharmaceutical composition that contains the compounds of this invention can be any formulation that is fit to predetermined medication, for example comprises solution, suspension or emulsion. Usually prepare solution, suspension and emulsion with the liquid embarkation body. Implement the used liquid embarkation body of the present invention and comprise, for example, water, salt solution, pharmaceutically acceptable organic solvent, pharmaceutically acceptable oil or fat etc., and two or more mixture wherein. The liquid embarkation body can contain other suitable pharmaceutically acceptable additive, such as solubilizer, emulsifying agent, nutrient, buffer, anticorrisive agent, suspending agent, thickener, viscosity modifier, stabilizing agent etc. Suitable organic solvent comprises, for example, and monohydric alcohol such as ethanol, and polyalcohol such as glycerine. Suitable oil comprises, for example, and soybean oil, peanut oil, olive oil, safflower oil, cottonseed wet goods. For parenteral, described carrier also can contain oleate, such as ethyl oleate, isopropyl myristate etc. Composition of the present invention can also be the forms such as particulate, microcapsules, liposome methods, and two or more mixture wherein.

Other additive comprises immunostimulant known in the art. Immunostimulatory oligonucleotide and polynucleotides are described in PCT WO 98/55495 and PCT WO 98/16247. U.S. Patent application No.2002/0164341 has described adjuvant and the non-Nuclec acid adjuvants that comprises non-methylated CpG dinucleotides (CpG ODN). U.S. Patent application No.2002/0197269 has described the composition that contains antigen, antigenicity CpG-ODN and polycationic polymer. Other immunostimulating additive that can use this area to narrate, for example, such as U.S. Patent No. 5,026,546; U.S. Patent No. 4,806,352; With U.S. Patent No. 5,026,543 is described.

Can use the controlled release induction system, such as matrix system or the erodable system that can control diffusion, for example be described in: " DIFFUSION CONTROLLED matrix system " (the 155-198 page or leaf) of Lee and " erodable system " (the 199-224 page or leaf) of Ron and Langer, publication is at " discussion of controlled drug delivery " (Treatise on Controlled Drug Delivery), A.Kydonieus compiles, Marcel Dekker, Inc., New York 1992. Described matrix can be for example, can cut (such as the protease cutting) by for example hydrolysis or enzyme in position or the biological degradable material of spontaneous degraded in the body. Described induction system can be for example natural generation or synthetic polymer or copolymer, for example with form of hydrogels. Example with the polymer that can cut connecting key comprises polyester, positive polyesters, polyanhydride, polysaccharide, poly-(phosphate), polyamide, polyurethane, poly-(iminocarbonic ester) and poly-(phosphine nitrile).

The compounds of this invention can contain the form of the unit dose formulations of pharmaceutically acceptable conventional non-toxic carrier, adjuvant and carrier, on demand in intestines, oral, parenteral, hypogloeeis, suction spraying, rectum or topical. For example, suitable mode of administration comprise oral, subcutaneous, transdermal, thoroughly in mucous membrane, iontophoresis, intravenous, the muscle, in the peritonaeum, interior, subcutaneous, the rectum of nose etc. Topical also comprises cutaneous penetration, such as percutaneous plaster or iontophoresis device. The term parenteral comprises in hypodermic injection, intravenous, the muscle, breastbone inner injection, or inculcates technology.

Injectable preparation, for example aseptic parenteral solution or oiliness suspension can be according to known technique made with suitable dispersant or wetting agent and suspending agent. The sterile injectable goods also can be aseptic parenteral solution or the suspensions with the acceptable diluent of nontoxic parenteral or solvent preparation, such as the solution with the 1,3-PD preparation. Operable acceptable carrier and solvent comprise water, Ringer's solution and isotonic sodium chlorrde solution. In addition, the conventional sterile non-volatile oils that adopts is as solvent or suspension media. For this purpose, the nonvolatile oil of any gentleness be can use, synthetic monoglyceride or diglyceride comprised. In addition, aliphatic acid such as oleic acid also can be used in the injectable goods.

The rectally suppository of medicine can mix medicine and make with suitable nonirritant excipient, excipient such as cocoa butter and polyethylene glycol, and they are solid at normal temperatures but are liquid under rectal temperature, therefore can melt the release medicine in rectum.

Be used for oral solid dosage forms and comprise capsule, tablet, pill, powder agent and granule. In this solid dosage forms, reactive compound can mix with at least a inert diluent (such as sucrose, lactose or starch). Usually, this formulation also can contain other material outside the inert diluent, for example, and lubricant such as dolomol. If capsule, tablet and pill can contain buffer in this formulation. In addition, tablet or pill also can have casing.

Can comprise pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir for oral liquid dosage form, wherein contain the normally used inert diluent in this area, such as water. This composition also can contain adjuvant, such as wetting agent, emulsifying agent and suspending agent, cyclodextrin and sweetener, flavor enhancement and flavouring agent.

When mentioning mode of administration, should emphasize that it is the combination that the therapeutic agent of coordinating effect can be provided, no matter the first and the second medicament give together or respectively. Therefore, these two kinds of medicaments can give together in single dose or give respectively with different time and interval.

The effective dose of the compounds of this invention generally includes any dosage that is enough to treat virus infections with detecting.

Successful treatment to object of the present invention can cause being subjected to sx↓ or the alleviation of medicine or the biological disorderly object that torments, in order to for example stop disease to further develop or prevent disease.

The amount that can mix with transport agent the active component that forms single formulation depends on the host that is treated with concrete mode of administration and different. Yet should be appreciated that, the given dose level that is used for any concrete patient will depend on various factors, comprising the seriousness of the activity of used particular compound, patient's age, body weight, health status, sex, diet situation, administration time, method of administration, drainage rate, ingredients and the disease specific for the treatment of. Definite a certain to the treatment effective dose under the stable condition easily by routine test, belong to common clinician's technical ability and judgement.

The compounds of this invention can liposome form administration. As known in the art, liposome is usually with phosphatide or the preparation of other lipid matter. Form liposome by single or multiple lift hydration liquid crystal being dispersed in aqueous medium. Can use on any nontoxic, physiology that can form liposome and can accept and metabolizable lipid. The composition of the present invention of liposome form also contains stabilizing agent, anticorrisive agent, excipient etc. except containing compound of the present invention. Preferred lipid is natural and synthetic phosphatide and phosphatid ylcholine (lecithin). The method that forms liposome is known in the art. Referring to for example, Prescott compiles, " cell biology method " (Mthods in Cell Biology), and the XIV volume, Academic Press, New York, N.W., p.33, with reference to following (1976).

Although SMIP compound of the present invention can be used as independent active agents and gives, they also can unite use with one or more other medicaments for the treatment of SARS. Unite other representative medicaments of using with the treatment virus infections with the compounds of this invention and comprise, for example, interferon, Ribavirin, gancyclovir etc.

When other active agents and the compounds of this invention are united when using, the consumption of other active agents usually can according to " doctor's desk reference " (PHYSICIANS ' DESK REFERENCE, PDR, the 53rd edition, 1999) the pointed therapeutic dose of a book, this book is included this paper list of references in, or according to those of ordinary skills until the treatment effective dose.

The compounds of this invention and other therapeutic activity medicament can be according to the maximum clinical dosage of recommending or with than the low dosage administration. The dosage level of reactive compound can change to obtain required therapeutic response according to seriousness and the reaction of method of administration, disease in the present composition. Described administering drug combinations can be used as separately composition or as the single formulation administration that contains two kinds of medicaments. When as administering drug combinations, this therapeutic agent can be mixed with the composition that separates that gives at same time or different time, and perhaps this therapeutic agent can be used as single composition and gives.

Adopt method as herein described or other method well known in the art to be not difficult to synthesize compound of the present invention.

Described compound can be inorganic or the organic acid salt form uses. These salt include but not limited to: acetate, oxalate, alginates, citrate, aspartate, benzoate, benzene sulfonate, bisulphate, butyrate, camphorate, camsilate, digluconate, cyclopentane propionate, lauryl sulfate, esilate, gluceptate, glycerophosphate, Hemisulphate, enanthate, caproate, fumarate, hydrochloride, hydrobromate, hydriodate, 2-isethionate, lactate, maleate, mesylate, nicotinate, 2-naphthalene sulfonate, oxalates, salicylate, pectate (pectinate), persulfate, 3-phenpropionate, picrate, pivalate, propionate, succinate tartrate, rhodanate, tosilate and hendecane hydrochlorate. Alkaline nitrogen-containing group can be quaternized by following reagent in addition, such as elementary alkyl halide, and for example methyl, ethyl, propyl group and butyl chloride compound, bromide and iodide; Dialkyl sulfide is such as dimethyl, diethyl, dibutyl and diamyl sulfide; Long-chain halide is such as decyl, lauryl, nutmeg base and hard ester group chloride, bromide and iodide; Aralkyl halide, such as benzyl and phenethyl bromide compound, etc. Thereby can obtain water-soluble or oil-soluble or water or oily dispersible product.

The example that can be used to form the acid of pharmaceutically acceptable acid-addition salts comprises inorganic acid, and example hydrochloric acid, sulfuric acid and phosphoric acid, and organic acid are such as oxalic acid, maleic acid, butanedioic acid and citric acid. Base addition salts can be in the final separation of the compound of formula (I) and the preparation of purification step situ, or its carboxylic moiety is prepared with suitable alkali (such as hydroxide, carbonate or the bicarbonate of pharmaceutically acceptable metal cation) or with ammonium or organic primary, secondary, reactive tertiary amine respectively. Pharmaceutically acceptable salt includes but not limited to: the cation of alkali metal or alkaline-earth metal, such as sodium, lithium, potassium, calcium, magnesium, aluminium salt etc., and nontoxic ammonium, quaternary ammonium and amine cation, include but not limited to: ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethamine etc. Other the representational organic amine that is used for forming base addition salts comprises diethylamine, ethylenediamine, monoethanolamine, diethanol amine, piperazine etc.

Various compounds and their synthetic methods are described in International Patent Application Publication No. WO02/18327 (based on the compound of benzamide and pyridinyl amides); WO0222598 and WO02/18383 (based on the compound of ABIQ); With WO 02/81443 (based on the compound of phthalamide (pthalamide)), these all within content of the present invention, can be used for strengthening immunity. These United States Patent (USP)s and international disclosed complete content are included this paper in as a reference. Interested other compound of the present invention or intermediate are used following methods available from commercial source: with interested chemical constitution drafting pattern input ACD-SC database (from MDL Information Systems). Search following company/research institute, compound supplier and purchase information: ASDI, ASINEX, BIONET, CHEMBRIDGE, CHEMDIV, CHEMEX, CHEMSTAR, COMGENEX, CSC, INTERBIOSCREEN, LABOTEST, MAYBRIDGE, MICROSOURCE/GENESIS, OLIVIA, ORION, PEAKDALE, RYAN SCIENTIFIC, SPECS, TIMTEC, U OF FLORIDA and the ZELINSKY of retrieval through identifying.

The benzazole compound

Flow process 1

The compound of the present invention that contains the benzimidazole core can prepare with the method that many these those skilled in the art are familiar with. In one approach, diamines and the various isothiocyanic acid ester coupling of suitable functionalization can be formed the thiocarbamide intermediate. Can be under known conditions, as processing with carbodiimide or alkyl halide, cyclisation forms the benzimidazole part. Perhaps can make diamines successively with the reaction of carbonyl dimidazoles and phosphoryl chloride phosphorus oxychloride, and then with suitable amine coupling.

Can be according to the method described above or other known conventional method prepare the compound of Han You oxazole structure. Haviv etc. (J.Med.Chem.1988,31,1719) have described a kind of method of Zhuan Pei oxazole core, and wherein hydroxyanilines is processed with ehtyl potassium xanthate. But then chlorination gained sulfonyl benzoxazole and with the amine coupling.

Contain the also available known method preparation of compound of benzothiazole core. Can make the reaction of ortho-, meta-or p-halo thiocarbimide ester and amine form thiocarbamide. Then reduce to form thiazole ring with NaH.

Benzothiazole replaces with method of the present invention usually, for example by following route of synthesis:

Synthetic 4-[(2-{[4-chloro-3-(trifluoromethyl) phenyl] amino }-

1H-benzimidazolyl-6-yl) oxygen]-N-picoline-2-carboxylic acid amides

Synthetic following compound 4-[(2-{[4-chloro-3-(trifluoromethyl) phenyl] amino }-1H-benzimidazolyl-6-yl) oxygen]-N-picoline-2-carboxylic acid amides (159322):

Step 1. is synthesized 4-[(4-amino-3-nitrobenzophenone) oxygen]-N-picoline-2-carboxylic acid amides: the mixture that will contain 4-amino-3-nitro phenol (1eq) and two (trimethyl silyl) acid amides potassium (2eq) stirred 2 hours under the room temperature in dimethyl formamide. Adding 90 ℃ in (4-chlorine (2-pyridine radicals))-N-methyl carboxylic acid amides (1eq) and potash (1.2eq) in this mixture stirred 3 days. Then concentrated reaction mixture distributes between ethyl acetate and water. Separate organic layer and obtain brown solid with salt water washing, drying, filtration and Vacuum Concentration. Obtain orange solids 4-[(4-amino-3-nitrobenzophenone by silica gel purification (2% triethylamine/50% ethyl acetate is dissolved in hexane)) oxygen]-N-picoline-2-carboxylic acid amides. This product has gratifying NMR. HPLC, 3.39min; MS:MH⁺=289。

Step 2. is synthesized 4-[(3, the 4-diamino-phenyl) oxygen]-N-picoline-2-carboxylic acid amides: the mixture that will contain [4-(3-amino-4-nitrophenoxy) (2-pyridine radicals)]-N-is dissolved in the 10%Pd/C hydrogenation that methyl alcohol is used catalytic amount, until yellow the disappearance obtains product amine. HPLC, 2.5mins; MS:MH⁺＝259。

Step 3. is synthesized 4-[(2-{[4-chloro-3-(trifluoromethyl) phenyl] amino }-1H-benzimidazolyl-6-yl) oxygen]-N-picoline-2-carboxylic acid amides:

To be dissolved in the 4-[(3 that contains of oxolane, the 4-diamino-phenyl) oxygen]-the mixture stirring at room of N-picoline-2-carboxylic acid amides (1eq) and 4-chloro-3-(trifluoromethyl) PhNCS (1eq) 16 hours to be to obtain corresponding thiocarbamide. Adding hydrochloric acid 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (2eq) in the gained mixture stirs mixture 10 hours again. Mixture is concentrated and distribute between ethyl acetate and water. Organic layer is also dry with the salt water washing. Obtain 4-[(2-{[4-chloro-3-(trifluoromethyl) phenyl by the HPLC purifying] amino }-1H-benzimidazolyl-6-yl) oxygen]-N-picoline-2-carboxylic acid amides. MS:MH⁺＝462

Synthetic 4-(the 2-[(4-bromophenyl) amino]-the 1-methyl-

1H-benzimidazolyl-5-yl } oxygen)-N-picoline-2-carboxylic acid amides

Synthetic following compound 4-(the 2-[(4-bromophenyl) amino]-1-methyl isophthalic acid H-benzimidazolyl-5-yl } oxygen)-N-picoline-2-carboxylic acid amides (161651):

Step 1. is synthesized 4-{[3-amino-4-(methylamino) phenyl] oxygen }-N-picoline-2-carboxylic acid amides: 4-[(4-amino-3-nitrobenzophenone) oxygen]-the METHYLENE CHLORIDE solution of N-picoline-2-carboxylic acid amides (1eq) processes with TFAA (1eq) and stirred 10 minutes at 0 ℃. Mixture is with saturated NaHCO₃The solution quencher. Separate organic layer and water, salt water washing, dry and evaporation. MS:MH⁺＝385.2

Add zephiran chloride trimethyl ammonium (1eq) dimethyl suflfate (1.2eq) in trifluoroacetamide (1eq) solution in toluene, acetonitrile and NaOH mixed liquor (50%). At room temperature stirring this biphase mixture spends the night and evaporates. Mixture is added in the ethyl acetate water, salt water washing, dry and evaporation. Thick product passes through column chromatography purification, with 1: 1 hexane and ethyl acetate, then 2% triethylamine (joining with 1: 1 hexane and ethyl acetate), 2% triethylamine (joining with 1: 1 hexane and ethyl acetate) wash-out then again, obtain orange red solid N-methyl-4-{[4-(methylamino)-3-nitrobenzophenone] oxygen } pyridine-2-carboxylic acid amides. MS:MH⁺＝303.1。

The methanol solution of nitro methylaniline is processed with 5% palladium/carbon and room temperature stirs 15 minutes (until yellow disappearance) under hydrogen. Filtering mixt, concentrated filtrate obtain 0.36 gram diamines 4-{[3-amino-4-(methylamino) phenyl] oxygen }-N-picoline-2-carboxylic acid amides. MS:MH⁺＝273.3。

Step 2. synthesize 4-((2-[(4-bromophenyl) amino]-1-methyl isophthalic acid H-benzimidazolyl-5-yl oxygen)-N-picoline-2-carboxylic acid amides: with diamines 4-{[3-amino-4-(methylamino) phenyl] oxygen-methanol solution of N-picoline-2-carboxylic acid amides (1eq) processes with 4-bromophenyl isothiocyanates (1eq) and stirred 2 hours at 60-65 ℃. Reactant mixture is cooled to room temperature, adds iodomethane (1eq) and spend the night 60 ℃ of stirrings. With reactant be cooled to room temperature, evaporate, pour into ethyl acetate, water and salt water washing, dry and reduction vaporization. Adopt gradient solvent system (hexane and ethyl acetate, and 1: 1 METHYLENE CHLORIDE of joining with METHYLENE CHLORIDE and acetone or 5% methyl alcohol) to carry out column chromatography, obtain the powdery product of half white. MS:MH⁺＝452.3

Aminobenzimidazole base quinolinone

The compound of structure I be from the simple starting molecule shown in the flow process 1-4 synthetic and exemplify in an embodiment. Shown in flow process 1, usually use the compound that is prepared structure I by the phenolic compound of amine and hydroxy-acid group replacement.

Flow process 2

Shown in flow process 2, substituted aromatic is as replacing or unsubstituted 2-amino benzoic Acid can produce acid amides with carboxylic acid halides such as the reaction of 2-(chloro carbonyl) methyl acetate, the gained acid amides will with replacement or unsubstituted 1, the reaction of 2-diaminobenzene. Products therefrom is the compound of the 4-hydroxyl-replacement of structure I. One skilled in the art will know that, can repair the listed method of calcium current journey 1, to make standby various compounds.

The method of the amino compound that replaces of 4-of preparation structure I is shown in flow process 3. Shown in flow process 3, can utilize amine and itrile group substituted aromatic to come the amino compound that replaces of 4-of composite structure I. Compound such as 2-cyan-acetic ester can produce hydrochloric acid 3-ethyoxyl-3-imino acetone acetoacetic ester with ethanol synthesis. With replacement or unsubstituted 1, the reaction of 2-phenylenediamine makes and replaces or unsubstituted 2-benzimidazolyl-2-guanidine-acetic acid ethyl ester subsequently. Replace or unsubstituted 2-benzimidazolyl-2-guanidine-acetic acid ethyl ester and the aromatic that contains amine and itrile group, as replacing or unsubstituted 2-aminobenzonitrile and alkali such as two (trimethyl silyl) acid amides lithium or lewis acid such as butter of tin react, obtain replacement or the amino compound that replaces of unsubstituted 4-of structure I.

Flow process 3

Flow process 4 shows the 4-dialkyl amido of composite structure I and the conventional route of synthesis of 4-amino compounds. Flow process 3 shows that the compound that the 4-hydroxyl of structure I replaces can be by changing into the 4-chlorine derivative with phosphorus oxychloride or thionyl chloride reaction. Then this 4-chlorine derivative can make corresponding 4-alkyl amino or 4-dialkyl amino radical derivative with alkylamine or dialkylamine reaction. Remove to protect the 4-alkyl amino or the 4-dialkyl amino based compound that obtain final structure I. Also can be by this way include but not limited to ROH, RSH and CuCN with other group of 4-chlorine derivative reaction.

Flow process 4

Shown in flow process 5, can synthesize the compound that has the structure I of H, alkyl, aryl or heterocyclic radical in the 4-position with replacement or the unsubstituted 2-benzimidazolyl-2-yl acetate by flow process 3 and 4 preparations.

Thiosemicarbazone (THIOSEMCARBAZONE)

The conventional method of preparation thiosemicarbazone

Flow process 6

The acetic acid solution stirring of aldehyde (1.0 equivalent) and thiosemicarbazone (1.05 equivalent) is spent the night. Remove excessive acetic acid and obtain residue, obtain thiosemicarbazone with the ethanol washing or by the preparation HPLC purifying.

Flow process 7

The methanol solution stirring of aldehyde (1.0 equivalent), thiosemicarbazone (1.05 equivalent) and acetic acid (0.1 equivalent) is spent the night. Remove methyl alcohol and obtain residue, this residue can be used for flow process 6.

Flow process 8

In the ethanolic solution of { [(1E)-1-azepine-2-(4-fluoro-3-nitrobenzophenone) vinyl] amino }-aminomethane-1-thioketones, add arylamine (2.1 equivalent). This solution at room temperature stirs until initial fluoride disappears. This solution purification is become product.

Flow process 9

With the mixture of 4-in the ethanol (diethylamino)-Benzaldehyde,2-hydroxy (1 equivalent), cylite (1.2 equivalent) and powder potash stirring at room 2 days. Remove ethanol, residue is dissolved in ethyl acetate and water. Organic layer NaHCO₃Na is used in the aqueous solution and salt water washing₂SO ₄Dry and concentrated. Residue obtains 4-(diethylamino)-2-benzoxy-benzaldehyde by silica gel purification with the ethyl acetate/hexane wash-out.

According to flow process 7 aldehyde is changed into thiosemicarbazone.

Flow process 10

The nmp solution of 3,4-difluorobenzonilyile (1 equivalent), amine (1.5 equivalent) and DIEA (2 equivalent) was heated 30 minutes in Smith micro-wave oven (Personal Chemistry). Reactant mixture obtains the 3-fluorobenzonitrile that 4-replaces by silica gel purification.

Under-78 ℃, in the toluene solution of nitrile, add DIBAL-H (1M is dissolved in toluene, 1.5 equivalents). Make reactant mixture be heated to room temperature, and stirred 16 hours, with methanol/ethyl acetate/salt solution (1: 1: 4) quencher. In stirring at room after 30 minutes, solution is with ethyl acetate extraction (3x). The organic layer NaHCO that merges₃The aqueous solution, salt water washing, and concentrated. Aldehyde is by silica gel purification or directly change into thiosemicarbazone (flow process 7).

Flow process 11

The THF solution of 2,4,5-trifluorobenzonitrile (1 equivalent) and 4-aryl piperazines (1.2 equivalent) and DIEA (1.2 equivalent) was heated 2 hours at 80 ℃. Mixture obtains 2 of 4-replacement, 5-difluorobenzonilyile by silica gel purification.

Flow process 12

THF (1M, the 1.1 equivalents) solution that in alcohol (1.0 equivalent), adds uncle-butoxy potassium. After 5 minutes, this solution is added-2 of 4-N-replacement, the THF solution of 5-difluorobenzonilyile (1 equivalent). Reactant mixture at room temperature stirs and spends the night and use the aqueous ammonium chloride solution quencher. Water-bearing layer ethyl acetate extraction (3x). The organic layer salt water washing that merges, and concentrated obtaining residue, this residue is purified obtain that 4-N-replaces-the 2-O-replacement-the 5-fluorobenzonitrile.

According to the step of flow process 10, with DIBAL-H reduction 4-N-replace-2-O-replaces-the 5-fluorobenzonitrile obtains that 4-N-replaces-2-O-replaces-the 5-fluorobenzaldehyde.

With flow process 7 this aldehyde is changed into corresponding thiosemicarbazone.

Flow process 13

With-2 of 4-N-replacement, the nmp solution of 5-difluorobenzonilyile (1 equivalent), amine (1.5 equivalent) and DIEA (2 equivalent) heated 30 minutes in Smith micro-wave oven (Personal Chemistry). Reactant mixture by silica gel purification with obtain that 4-N-replaces-2-N-replaces-the 5-fluorobenzonitrile.

According to the step of flow process 10, with DIBAL-H reduction 4-N-replace-2-N-replaces-the 5-fluorobenzonitrile obtains that 4-N-replaces-2-N-replaces-the 5-fluorobenzaldehyde.

Preparation amino 3-[5-(3-chlorphenyl) (2-furyl)] (2-pyrazolinyl) } methane-1-thioketones

Under 0 ℃, in the THF solution of 5-(3-chlorphenyl) furans-2-carbon aldehyde (5-(3-chlorophenyl) furan-2-carbaldehyde) (1.0 equivalent), add ether-soluble MeMgBr (3.0 equivalent), and stirred 45 minutes. The quencher of reactant water is filtered with the ether dilution and by diatomite (Celite). Organic layer is separated and use the salt water washing, pass through MgSO₄Drying, and concentrated 1-[5-(3-the chlorphenyl)-2-furyl that obtains] second-1-alcohol.

CH at secondary alcohol (1.0 equivalent)₂Cl ₂Add MnO in the solution₂(10 equivalent). The reactant stirring is spent the night, filter by Celite, and concentrated 1-[5-(3-the chlorphenyl)-2-furyl that obtains] second-1-ketone.

In the alcohol mixeding liquid of ketone (1.0 equivalent), paraformaldehyde (2.0 equivalent) and dimethylamine hydrochloride (2.0 equivalent) and molecular sieve, add concentrated hydrochloric acid (cat.). Reactant spends the night and concentrated in refluxed under nitrogen. Add several HCl, mixture is prepared with DCM and water. Discard organic layer. The water-bearing layer is transferred to alkalescence and extracts (3x) with DCM. MgSO is passed through in organic layer salt water washing₄Drying, and concentrated 3-(the dimethylamino)-1-[5-(3-chlorphenyl) (2-furyl) that obtains] third-1-ketone.

Thiosemicarbazone (1.0 equivalent) is dissolved in MeOH, under nitrogen, heats simultaneously. In reactant, add sodium hydrate aqueous solution (6M, 9.0 equivalents). In reactant mixture, dropwise add 3-(dimethylamino)-1-[5-(3-chlorphenyl) (2-furyl)] methanol solution of third-1-ketone (1.0 equivalent). Desolventizing is dissolved in DCM and water, salt water washing with residue, passes through MgSO₄Drying, and concentrated. Final compound obtains amino { 3-[5-(3-chlorphenyl) (2-furyl)] (2-pyrazolinyl) } methane-1-thioketones by the preparation HPLC purifying; LC/MS m/z 306.2 (MH+); Rt=3.06 minute.

Flow process 14

CHCl at 4-pyridine radicals methylamine (1.0 equivalent) and triethylamine (2.0 equivalent)₃Add CS in the solution₂(1.0 equivalent)) and stir and spend the night. Reactant is cooled to 0 ℃ also dropwise adds ethyl chloroformate (1.0 equivalent). Reactant stirred 15 minutes at 0 ℃, then at room temperature stirred 2 hours, added (tert-butyl) oxygen carbohydrazide (1.2 equivalent) again. Stir again after 1 hour with aqueous citric acid solution (5%), saturated NaHCO₃With the salt solution purging compound, pass through MgSO₄Drying, and concentrated. Thiosemicarbazone with the required Boc protection of column chromatography purification.

In thiosemicarbazone (1.0 equivalent) solution of the Boc protection that is dissolved in DCM, add HCl diox (2M, 8.3 equivalents) solution, and stirred 15 minutes. Then add MeOH with dissolution precipitation, add again alditol and a small amount of acetic acid (0.5 mL). Spending the night the mixture stirring also, desolventizing obtains thiosemicarbazone to obtain residue by preparation type-HPLC purifying.

Synthetic 4-[4-(4-methylpiperazine-1-yl) phenoxymethyl] benzaldehyde

At the CHCl that is cooled to 0 ℃ 4-piperazine-1-base phenol (1 equivalent)₃The CHCl that dropwise adds dimethyl dicarbonate butyl ester (1 equivalent) in the solution₃Solution. Solution stirred 1 hour at 0 ℃, then took out from cryostat and at room temperature stirred 18 hours. Organic solution NaHCO₃MgSO is passed through in the aqueous solution and salt water washing₄Dry and concentrated, thick material need not purifying and can use.

Under the room temperature nitrogen, at the anhydrous CH of gained 4-(1-BOC-piperazine-4-yl) phenol (1 equivalent)₃Dropwise add lentamente anhydrous CH in the CN solution₃NaH among the CN (1 equivalent) slurries. These slurries were at room temperature stirred 2 hours, then cross filter solid and use Et₂The O washing.

In anhydrous propanone, mix 4-(1-BOC-piperazine-4-yl) sodium phenate (1 equivalent) and added hot reflux 18 hours with 4-bromomethyl-benzoic acid methyl ester (1 equivalent) and at 60 ℃. With dope filtration, then concentrated filtrate is to obtain rough 4-[4-(1-BOC-piperazine-4-yl) phenoxymethyl] methyl benzoate, this material need not purifying and can use.

Under the nitrogen, the LiAlH in being cooled to 0 ℃ anhydrous THF₄Dropwise add lentamente 4-[4-(1-BOC-piperazine-4-yl) phenoxymethyl in (4 equivalent) slurries] anhydrous THF solution of methyl benzoate (1 equivalent). In case add fully, these slurries added hot reflux 1 hour at 80 ℃. Also water, the 10%NaOH aqueous solution are processed, and then are used water treatment subsequently these slurries to be cooled to 0 ℃. With the gained solid filtering, filtrate is diluted with chloroform, uses the salt water washing, passes through MgSO₄Dry and concentrated, obtain rough 4-[4-(4-methylpiperazine-1-yl) phenoxymethyl] phenmethylol, need not purifying can use.

Under the nitrogen, be cooled in-78 ℃ the anhydrous DCM solution of DMSO (2.6 equivalent) and dropwise adding the oxalyl chloride (1.1 equivalent) that is dissolved in DCM. Solution stirred 5 minutes at-78 ℃, then dropwise added 4-[4-(4-methyl piperazine-1-yl) phenoxymethyl] the DCM solution of phenmethylol (1 equivalent), and again-78 ℃ of stirrings 30 minutes. Slowly add triethylamine (2.5 equivalent), then make solution reach room temperature. Solution NaHCO₃MgSO is passed through in the aqueous solution and salt water washing₄Drying, and concentrated rough 4-[4-(4-methylpiperazine-1-yl) phenoxymethyl that obtains] benzaldehyde, convert it into thiosemicarbazone according to flow process 7.

The pyrroles

Flow process 15

The pyrroles's is synthetic

Preparation (2E)-3-(2,4-dichlorophenyl) third-2-olefin(e) acid tert-butyl ester (2).

Under the room temperature argon gas, at well-beaten cinnamate (1eq), the tert-butyl alcohol (4eq), DMAP (1.4eq) and CH₂Cl ₂Solution in add pure DIC (1.4eq). (attention-cinnamate must be dissolved in solution fully, therefore may need slightly to heat. Make solution be cooled to room temperature, then add DIC. For avoiding fairly large heat release, can use first CH₂Cl ₂Careful dilution DIC adds again, and prepares an ice bath. ) stir after 8 hours, form white precipitate in the reactant. Can pass through the TLC detection reaction, with the 25%EtOAc/ hexane wash-out (R of product_fBe 0.9). All reactant transfer (are used CH to separatory funnel₂Cl ₂Washing). Organic mixture citrate, saturated NaHCO₃The aqueous solution, water and salt water washing. With the dry (Na of organic layer₂SO ₄), filter, and be concentrated into dried to obtain oily crude product. Rough grease is mixed with hexane and stirred 30 minutes. The precipitation that forms is filtered evaporated filtrate by celite. Hexanes mixtures is transferred on the silica filter plug core with EtOAc/ hexane (97: 2v/v) wash-out. The UV active component of collecting the wash-out first time is also evaporated to obtain 2 (productive rate 75-80%) of purity＞99%.

Preparation 4-(2,4-dichlorophenyl) pyrroles-3-carboxylic acid tert-butyl ester (3).

In NaH (1.5eq, oiliness decentralized photo), add absolute ether under the argon gas. Remove ether by the syringe decant, under argon gas, make again NaH be suspended in fresh ether. Under 0 ℃, dropwise join the NaH suspension of stirring with the solution of the TOSMIC (1.1eq) that will be dissolved in ether and DMSO mixture in 20-30 minute and 2 (1eq). Slightly heat release and have gas to generate of adition process. After the adding, make reactant reply room temperature. Then reactant is carried out TLC (25%EtOAc/ hexane, the R of UV activated product_f=0.4) and LCMS, until react completely (about 2-3 hour). After reaction finishes, with saturated NH₄The careful quencher reactant of the Cl aqueous solution (slowly adding to avoid strong gas to occur and heat release) also dilutes with ether. Separate each layer, the saturated NaHCO of organic phase₃The aqueous solution, water and salt water washing. Rough black solid can pass through recrystallization purifying. By (1: 3v/v) mixture directly is recrystallized or then the EtOAc that crude product is dissolved in low-grade fever is added hexane (volume of hexane is about the twice of EtOAc volume) and can obtain optimum from the EtOAc/ hexane of heat. Then make hot solution be cooled to room temperature and age overnight. At first crystallization is filtered, then with the hexane washing, obtain 99% pure product, productive rate is 60-70%.

Preparation 4-(2,4-dichlorophenyl)-1-[3-(1,3-Er Evil benzo [c] azoles quinoline-2-yl) propyl group] pyrroles-3-carboxylic acid tert-butyl ester (4).

Room temperature is with under the argon gas, adds solid NaH (1.5eq, oiliness decentralized photo) while stirring in fraction is dissolved in the solution of the pyrroles 3 (1eq) of DMF and 3-bromopropyl phthalimide (1.2eq). Notice-have that some gases generate, but as if temperature can not raise above 40-50 ℃. Reactant was stirred 1.5 hours under the room temperature argon gas. Then carry out TLC (CH₂Cl ₂/ acetonitrile (95: 5v/v), the R of UV activated product_f=0.5) and LCMS. After reaction finishes, with saturated NH₄The careful quencher reactant of the Cl aqueous solution (slowly adding to avoid strong gas to occur and heat release). Then add saturated NaHCO₃The aqueous solution to be avoiding emulsification, and extracts alkaline organic mixture with ether. The ether layer that merges is with saturated NaHCO₃Na is passed through in the aqueous solution, water, salt water washing₂SO ₄Drying is filtered, and is concentrated into dried to obtain thick product. Thick product is by silica EtOAc/ hexane (1: 4v/v) wash-out purifying. Contain some remaining 3-bromopropyl phthalimides in the product of purifying, it can not disturb synthesis step subsequently. This material is taken out, and it need not be further purified just and can use. Suppose that productive rate is certain.

Preparation 1-(3-aminopropyl)-4-(2,4-dichlorophenyl) pyrroles-3-carboxylic acid tert-butyl ester (5).

Under the room temperature nitrogen phthaloyl imino pyrroles (pthalimido pyrroles o) 4 (1eq) are dissolved in ethanol and hydrazine (3eq). Add the hot reflux afterreaction and form white precipitate. Reactant is carried out TLC (CH₂Cl ₂/ acetonitrile (95: 5v/v), the R of UV activated product_f=0.2) and the LCMS return stirring until react completely (about 2 hours) judge. After reaction finishes, make reactant be cooled to room temperature, and with the thin sintered glass filter vacuum of pore elimination medium. Filtrate decompression is condensed into the stickiness solid. Crude product is placed ethanol/EtOAc (1: 1v/v), stir and use and above-mentioned same mode elimination precipitation. Filtrate decompression is concentrated, then under vacuum dry 10-15 minute. Add ethanol/EtOAc, filtration and concentrated process and be repeated one or many, or carry out as required, to remove most of white precipitates and remaining hydrazine. Product is dried overnight under vacuum then. The gained material need not to be further purified and can use. In case dry, this reactant produces glass product (productive rate of 2 steps about 87%).

Preparation 1-{3-[(6-amino-5-nitro (2-pyridine radicals)) amino] propyl group }-4-(2,4-dichlorophenyl) pyrroles-3-carboxylic acid tert-butyl ester (7).

In premixed pyrroles 5 (1eq) and pulverous 6-chloro-3-nitro-2-pyridine radicals amine (6) anhydrous reagent (1.1eq), add DMA, then add H ü nig alkali (2eq), at room temperature stir subsequently. Then reactant being heated to 80 ℃ spends the night. Then to reactant carry out TLC (the EtOAc/ hexane (and 1: 1v/v), the R of UV active yellow product_f=0.25), HPLC and LCMS. Judge by HPLC and to make reactant be cooled to 70 ℃ after reacting completely. Then in reactant, add ethylenediamine (anhydrous) to destroy the unreacted chloropyridine 6 of any remnants. 70 ℃ stir 15 minutes after, with the reactant cooling and add saturated NaHCO₃Aqueous solution quencher. Mixture aqueous solution extracts with EtOAc, and the organic layer of merging is with saturated NaHCO₃The aqueous solution, water, salt water washing, drying is filtered, and is concentrated into dried to obtain the thick product of brown color solid shape. Thick product is by the flash distillation chromatographic purifying, with EtOAc/ hexane (4: 6v/v) wash-out. Separate the SnAr adduct 7 that obtains purifying, productive rate 58%, it is yellow solid.

Preparation 1-{3-[(6-amino-5-nitro (2-pyridine radicals)) amino] propyl group }-4-(2,4-dichlorophenyl) pyrroles-3-carboxylic acid (8).

Under the room temperature, pyrroles's tert-butyl ester 7 (1eq), water (.1%) and CH in test tube₂Cl ₂Mixture in add while stirring TFA (catalytic amount). At room temperature stir test tube until reaction finishes (about 12 hours). Then reduced pressure concentration reactant and dry under vacuum at room temperature. Rough residue is dissolved in CH again₂Cl ₂And reduced pressure concentration at room temperature. The gained material need not to be further purified and just can be used as tfa salt for final coupling step.

Preparation N-((1S)-2-hydroxyl-isopropyl) (1-{3-[(6-amino-5-nitro (2-pyridine radicals)) amino] propyl group }-4-(2,4-dichlorophenyl) pyrroles-3-yl) carboxylic acid amides (9).

Under the room temperature argon gas, acid (8) (1eq), adding (2S)-(+)-2-aminopropan-1-ols (1.5 eq) while stirring in the mixture of HBTU (1.5eq), H ü nig alkali (2eq) and DMF (according to this successively premixed in test tube of order). Reactant was stirred 3-4 hour until show that by LCMS and HPLC reaction finishes. Reactant mixture with the EtOAc dilution, is used NaHCO subsequently₃Washing is also concentrated to obtain a kind of powder, and productive rate is 70%.

Use the Development available from Advanced Chemistry, the compound among ACD Name software (version 5.07,2001/11/14) the name embodiment of Inc. Some compounds and initial substance are named with standard I UPAC nomenclature.

Compound in the table 34 is synthetic with the synthetic method described in above-described embodiment and the flow process, and 1 and 2 screens according to the methods below. Used precursor is that the technical staff who is proficient in this field is convenient to understand, and can be available from Aldrich (Milwaukee, WI) or Acros Organics (Pittsburgh, PA).

The screening technique of SMIP/SMIS compound

Method 1

Can be at the little molecular immune reinforcing agent of external evaluation candidate. Ability according to the compound immune cell activated is screened external. A kind of mark of this activity is to induce to produce cell factor (for example producing TNF-α). Apoptosis-induced little molecule is accredited as has this activity. These little molecular immune reinforcing agents have the potential use as adjuvant and immunotherapy.

In the mensuration process (high flux screening (HTS)) of little molecular immune reinforcing agent (SMIP), with human peripheral blood single nucleus cell (PBMC) (500,000/mL, cultivate basigamy with the RPMI 1640 that is added with 10%FCS) add and contained 5 μ M with in 96 orifice plates of the compound of DMSO preparation (every hole 100,000). With PBMC at 37 ℃, 5%CO₂Lower cultivation 18 hours. Measure they produce cell factor in to the micromolecular compound reaction ability with improved sandwich ELISA.

In brief, adopt the flat board be combined with the first antibody of catching usefulness, then form with the SA of biotinylation anti-TNF sandwich, with the TNF that secretes in the mensuration PBMC culture supernatant. Then with the SA of Avidin-europium detection of biological elementization, use the binding capacity of time-resolved fluorometry europium. Confirm the TNF induced activity of SMIP compound in this experiment, this activity shows as the Europim counting to be increased than the cell of only cultivating in the RPMI culture medium. Select " person of hitting " according to them with respect to the TNF induced activity of the strong derivant lipopolysaccharides of TNF LPS (1 μ g/ml) optimal dose. The robustness of this mensuration and low background be selected the compound that hits with about 10%LPS activity so that energy is conventional, are generally 5-10 times (only cell) of background. Then need to confirm selected hit compound and when low concentration, can induce the multidigit blood donor to produce cell factor. Those compounds that have a constant activity at 5 μ M or lower concentration are considered to meet the purpose of this mensuration. Easily this test is improved to screen compounds effective when higher or the low concentration.

Method 2

Every kind of compound in the table 34 can both induce human peripheral blood single nucleus cell to produce TNF-α. Wherein chemical compound lot shows the activity of inducing generation TNF-α when being lower than 20 μ M. Chemical compound lot has shown when being lower than 5 μ M induces the activity that produces TNF-α. Chemical compound lot shows when being lower than 1.5 μ M induces the activity that produces TNF-α.

For this reason, each R group of listed any compound is preferred in the table 34. In addition, because the outstanding activity of every kind of compound, in these compounds each is preferred, and the member in the preferred group as comprising any or all other compound, and preferred every kind of compound in regulating the immunity method that strengthens and the method for the treatment of the biological illness relevant with it for example can be used as vaccine adjuvant. Also preferably make for vaccine, the immune medicine that strengthens, reduces tumor growth and treat its biological illness that causes with every kind of compound. Except said method, the method for measuring other cell factor (such as IL1-β, IL-12, IL-6, IFN-γ, IL-10 etc.) is well known in the art, can be used to seek active SMIP compound of the present invention.

Can adopt can be at higher concentration in above-mentioned test, the compound that causes TNF-α to produce during such as 100 μ M, 200 μ M or 300 μ M. For example, Loxoribine (Loxoribine) can cause effectively producing TNF-α (referring to Pope etc. at 300 μ M, immune-stimulating compound 7-pi-allyl-8-oxygen guanosine (Loxoribine) is induced significant mouse cellular immunity cell factor subgroup (Immunostimulatory Compound 7-Allyl-8-Oxoguanosine (Loxoribine) Induces a Distinct Subset of Murine Cytokines Cellular Immunology), 162:333-339 (1995)).

The present invention also comprises isotope-labeled antiviral compound, its structure and above-mentioned those Compound Phases with, but have one or more atoms to be different from atomic weight that occurring in nature finds usually by atomic weight or mass number or the atom of mass number substitutes. The isotopic example that can be impregnated in antiviral compound of the present invention comprises the isotope of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine, respectively as²H、 ³H、 ¹³C、 ¹⁴C、 ¹⁵N、 ¹⁸O、 ¹⁷O、 ³¹P、 ³²P、 ³⁵S、 ¹⁸F and³⁶Cl. The pharmaceutically acceptable salt that contains other isotopic antiviral compound of the present invention, its derivative, described compound and the described derivative of above-mentioned isotope and/or other atom is included within the scope of the invention. Some isotope-labeled antiviral compound of the present invention, for example wherein mix radio isotope as³H and¹⁴Those compounds of C are useful in medicine and/or substrate Tissue distribution mensuration. Because tritium (namely³H) and carbon-14 (namely¹⁴C) the easy preparation and determination methods of isotope is so be particularly preferred. In addition, with heavier isotope such as deuterium (namely²H) replacement may provide owing to higher metabolic stability some treatment benefit, as prolonging Half-life in vivo or reducing the dosage demand, is preferred in some cases therefore. Isotope-labeled antiviral compound of the present invention and derivative thereof can prepare with the method for mentioning in known method or the list of references usually, and can substitute the heterotope labelled reagent with not un-come-at-able isotope-labeled reagent.

According to the present invention, provide the SMIP compound of the using effective dose method as adjuvant. The immunogenic composition that contains SMIP compound, antigen and optional other adjuvant also is provided.

As adjuvant, SMIP compound and antigen and induction system can be made up to form final immunogenic composition or vaccine product.

As immunotherapy, the SMIP compound can use separately or be used in combination with the other therapies for the treatment of SARS.

One of ordinary skill in the art will know, having can be near the physiologically active antiviral compound SMIP of hydroxyl or SMIS usually with the form administration of pharmaceutically acceptable ester. Antiviral compound of the present invention can be used as the effective administration of ester that its hydroxyl forms, as pharmaceutical chemical technical staff envision. The pharmaceutical chemistry field is known already, can regulate speed and the duration that antiviral compound is had an effect by selecting suitable ester group to be used for.

Can comprise with other compound that therapeutic combination as herein described uses, PTX (PTX), medrat, Trimetrexate (Neutrexin), Zadaxin (thymosin alpha 1), the optional 5-guanidine base (aminomethinimino) that replaces-3-methyl-4-isoxazole carboxylic acid phenyl acid amides, Ciclosporin A (CsA), 6-oxygen-1,4,5-thiazine [2,3-b] amino-2 (1H)-sulphur oxygen-4 (the 3H)-quinazolinones of quinazoline, 3-, gangciclovir, glycyrrhizin, Tetracyclines, aminoglycosides, quinolone, bicyclam (1,4-two (1,4,8,11-, four a word used for translation rings, four decane-1-ylmethyl) benzene eight hydrochloride dihydrate compounds), rapamycin, wortmannin, Enalapril, roquinimex/linomide, inactivin, DNCB, AG7088,9-aminocamptothecin (CPT-11), loxorobine, Bropirimine, Ononase^_(ranpirnase), statins (as: Lovastatin--Mevacor^_, Pravastatin--Pravachol^_, Simvastatin--Zocor^_, Fluvastatin--Lescol^_, Atorvastatin-Lipitor^_And rosuvastatin--Crestor^_)。

Term " effective dose " represents to treat the amount of antiviral compound in composition of the present invention, kit and the method for described disease here. Certainly, the concrete dosage that gives the compounds of this invention will be decided by concrete condition, comprising, for example, the compound of using, method of administration, patient's situation and the seriousness that is treated symptom.

The dosage that gives the antiviral compound of the present invention of object is can be different, and depends on clinician's judgement. It should be noted, when using with the form of salt (such as laruate), be necessary to regulate the dosage of compound, because salt can form certain the change of molecular weight.

Other dosage that following dosage and this specification other parts are mentioned is that body weight is about 65-70kg for each person. Whether the doctor who is proficient in can exist some disease (such as diabetes) to determine the object required dosage of body weight beyond the 65-70kg scope according to medical history and the object of object. The calculating of other form drug dose of free alkali form (such as salt or hydrate) can be not difficult to finish by the molecular weight of related medicament categories is made simple ratio.

Usually, pharmaceutical composition will comprise at least a and pharmaceutically acceptable carrier, such as salt solution, BS, the 5% dextrose aqueous solution, contain the BBS of trace metal etc., the antiviral compound of combination. Preparation also can comprise one or more excipient, anticorrisive agent, solubilizer, buffer, lubricant, filler, stabilizing agent etc. The preparation method is well known in the art and is described in, for example, " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences), Mack Pub.Co., New Jersey (1991) or " Lei Mingdun pharmaceutical science with put into practice " (Remington:The Science and Practice of Pharmacy), the 20th edition, Lippincott Williams ﹠ Wilkins, Baltimore, the Maryland State (2000) include this paper in as a reference.

Be used for pharmaceutical composition of the present invention and can be aseptic apyrogeneity liquid solution or suspension, dressing capsule, suppository, freeze-dried powder, transdermal pastes form, or other form known in the art.

Known many active component antiviral compounds absorb from alimentary canal, therefore, for easy reason, common preferred oral compound. Yet, if necessary, by intravenous, subcutaneous, through skin or absorb suppository as rectum or vagina to give compound effective too. The composition of all general types be can use, tablet, chewable tablets, capsule, solution, parenteral solution, lozenge, suppository and suspending agent comprised. Composition is made into to contain the part that makes things convenient for of daily dose or daily dose, takes dosage unit form, can be a slice medicine or capsule, or makes things convenient for the liquid of volume.

Capsule is to incapsulate preparation with one or more compounds and suitable diluent mixture and with an amount of mixture. Diluent commonly used comprises the inert powder material, such as many dissimilar starch, cellulose powder (especially avicel cellulose and microcrystalline cellulose), carbohydrate (such as fructose, sweet mellow wine and sucrose), cereal powder and similar edible powder.

Tablet is by direct pressing, by wet granulation or non-slurry pelletizing manufacturing. Generally include diluent, adhesive, lubricant and disintegrant in its prescription, and one or more compounds. Typically diluent comprises, for example, and various types of starch, lactose, sweet mellow wine, kaolin, calcium phosphate or calcium sulfate, inorganic salts (such as sodium chloride) and Icing Sugar. Also can use the cellulose derivative powder. Typical tablet binder has starch, gelatin and carbohydrate (such as lactose, fructose, glucose etc.). Also natural and synthetic colloid be can use, Arabic gum, alginates, methylcellulose, polyvinylpyrrolidine etc. comprised. Polyethylene glycol, ethyl cellulose and wax also usually are used as adhesive.

Usually need lubricant to adhere on the mould to prevent tablet and drift in the tablet formulation. Lubricant is selected from smooth solid, such as talcum, dolomol and calcium stearate, stearic acid and hydrogenated vegetable oil.

Tablet disintegrant is to expand the material that destroys tablet and discharge one or more compounds when moistening. They comprise starch, clay, cellulose, phycocolloid and natural gum, for example corn and farina, methylcellulose, agar, bentonite, lignose, natural sponge powder, cationic ion-exchange resin, alginic acid, guar gum, oranges and tangerines pulp and carboxymethyl cellulose are more specifically arranged, also can use NaLS.

Tablet also often is used as the sweet tablet of spices and sealer, or forms the protective agent dressing to change the dissolution characteristics of tablet with film. Described compound also can be made into chewable tablets, and this need to use the joyful material of relatively a large amount of tastes in prescription, and such as sweet mellow wine, this is now very ripe in this area.

When needs during with the suppository administered compound, can use typical matrix. Cocoa butter is a kind of traditional suppository base, can add wax and modify its fusing point that slightly raises. The suppository base that Yi Yushui mixes is widely used, and wherein especially comprises the polyethylene glycol of various molecular weight.

Can postpone or prolong function of chemical compound by suitable prescription. For example, can prepare the compound piller of slow dissolving and it is mixed tablet or capsule. This technology also can be modified, and namely makes the different piller of some dissolution rates and the mixture of these pillers is filled in the capsule. Tablet or capsule can be with resisting the film coating that dissolves in the scheduled time. Even the parenteral goods also can be made into long-acting form, method is with one or more compound dissolvings or is suspended in oiliness or the emulsification carrier that can slowly disperse in serum.

Drug regimen of the present invention can be used by controlled release preparation (for example slowly-releasing or quick-release formulation). This controlled release preparation of drug regimen of the present invention can prepare with the method known by the technical staff of being proficient in this field. The technical staff that medication will be proficient in this field by clinician or other decides after judging according to patient's illness and demand.

Term " prodrug " refers to transform in vivo the compound that (as by being hydrolyzed) forms antiviral compound of the present invention in blood. Conversion can occur by various mechanism, as by being hydrolyzed in blood. Be provided at T.Higuchi and W.Stella about being described in detail of prodrug, " as the prodrug of novel induction system " (Pro-drugs as Novel Delivery Systems), A.C.S. serial Conference Papers collection the 14th volume, and " the reversible carrier of the biology in the drug design " (Bioreversible Carriers in Drug Design), Edward B.Roche compiles, American Pharmaceutical Association Pergamon Press, 1987. Term " prodrug " comprises that also one or more antiviral compounds in the mutual prodrug are combined into single molecule, and this molecule can produce various antiviral compounds of the present invention through transforming.

For example, if antiviral compound of the present invention contains carboxylic acid functional, then prodrug can comprise the ester that replaces carboxylic acid group's hydrogen atom to form with following group, and these groups are (C for example₁-C ₈) alkyl, (C₂-C ₁₂) alkanoyloxymethyl, contain 4-9 carbon atom 1-(alkanoyloxy) ethyl, contain the 1-methyl isophthalic acid of 5-10 carbon atom-(alkanoyloxy)-ethyl, contain 3-6 carbon atom the alkoxyl carbonyl oxy-methyl, contain 4-7 carbon atom 1-(alkoxyl carbonyl oxygen base) ethyl, contain the 1-methyl isophthalic acid of 5-8 carbon atom-(alkoxyl carbonyl oxygen base) ethyl, N-(alkoxy carbonyl) amino methyl that contains 3-9 carbon atom, 1-(N-(alkoxy carbonyl) amino) ethyl that contains 4-10 carbon atom, 3-phenylpropyl alcohol [c] furanonyl, 4-crotonocyl lactone group (4-crotonolactonyl), γ-butyryl lactone-4-base, two-N, N-(C₁-C ₂) alkyl amino (C₂-C ₃) alkyl (such as β-dimethylaminoethyl), carbamyl-(C₁-C ₂) alkyl, N, N-two (C₁-C ₂) alkylcarbamoyl group-(C₁-C ₂) alkyl and pyrido-, pyrrolidines also-or morpholino (C₂-C ₃) alkyl.

Similarly, if antiviral compound of the present invention contains alcohol functional group, then can be by replacing the hydrogen atom of alcohol groups to form prodrug with following group, these groups are (C for example₁-C ₆) alkanoyloxymethyl, 1-((C₁-C ₆) alkanoyloxy) ethyl, 1-methyl isophthalic acid-((C₁-C ₆) alkanoyloxy) ethyl, (C₁-C ₆) alkoxyl carbonyl oxy-methyl, N-(C₁-C ₆) alkoxycarbonyl amino methyl, succinyl group, (C₁-C ₆) enol base, alpha-amido (C₁-C ₄) enol base, aryl-acyl and α-aminoacyl or alpha-amido acyl-alpha--aminoacyl, wherein each alpha-amido acyl group independently be selected from natural generation L-amino acid, P (O) (OH)₂、-P(O)(O(C ₁-C ₆) alkyl)₂Or glycosyl (removing the residue that produces behind the hydroxyl in the carbohydrate hemiacetal form).

If antiviral compound of the present invention contains amine functional group, then can be by replacing the hydrogen atom of amine groups to form prodrug with following group, these groups are R for example^X-carbonyl, R^XO-carbonyl, NR^XR ^X1-carbonyl, wherein R^XAnd R^X1Independent respectively is ((C₁-C ₁₀) alkyl, (C₃-C ₇) cycloalkyl, benzyl, or R^X-carbonyl is natural alpha-amido acyl group or natural alpha-amido acyl group-natural alpha-amido acyl group ,-C (OH) C (O) OY^X(Y wherein^XH, (C₁-C ₆) alkyl or benzyl) ,-C (OY^X0)Y ^X1, Y wherein^X0(C₁-C ₄) alkyl, Y^X1((C₁-C ₆) alkyl, carboxyl (C₁-C ₆) alkyl, amino (C₁-C ₄) alkyl or list-N-or two-N, N-(C₁-C ₆) the alkyl amino alkyl ,-C (Y^X2)Y ^X3, Y wherein^X2H or methyl, Y^X3List-N-or two-N, N-(C₁-C ₆) alkyl amino, morpholino, piperidin-1-yl or pyrrolidin-1-yl.

Composition can be prepared with conventional method with one or more physiologically acceptable carriers or excipient used according to the present invention. According to the United States Patent (USP) of listing in the description here and table 1, table 2, table 34 and the table 38 and the preparation of the description in the disclosed international patent application or obtained antiviral, SMIP, SMIS or other immunomodulatory compounds. Described antiviral compound can be formulated in and be fit to be transported in the pharmaceutically acceptable composition of lung. Concrete preparation comprises and is suitable as the spray used in the metered dose inhaler and dry powder, liquid solution or the suspension of propellant. The preparation of this preparation is be proficient in this field known by the technical staff, and is described in United States Patent (USP) 5,814,607 and 5,654,007 and table 3 in the United States Patent (USP) and disclosed international patent application listed, they are included into this paper as a reference.

Dry powder formulations will contain the antiviral compound of drying (optional is lyophilized form), within the scope that its granular size should can deposit in lung. Supply the particle size range of deposition in the lung usually between 1-5 μ m. In the time need to carrying antiviral compound with the mode whole body that enters blood flow from the lung absorption, the granularity of described antiviral compound preparation is usually between 0.1 and 2 μ m. Preferred particle size range can be with producing such as spraying the methods such as pulverizing, spray-drying and solvent deposition. The dry powder device usually require powder quality between about 1mg-100mg to produce aerosolizable dosage. Therefore, described antiviral compound will common and pharmaceutically acceptable dry powder blend in bulk. Preferred dry powder in bulk comprises sucrose, lactose, trehalose, human serum albumins (HSA), phosphatide and glycine, and described in the document listed of table 3 those. Dry powder can be administered to object with the Diskus of routine. For liquid preparation, described antiviral compound dissolves in the physiologically acceptable carrier that aerosolizable preparation is carried in any known being used for. This carrier comprises the aqueous solution of buffering and the water soluble compound that does not cushion, and physiological solution, comprising salt solution (preferred 0.2-2N NaCl). If the solubility of antiviral compound is limited, also can use other liquid embarkation body, such as ethanol, propane diols and ethanol-propylene compositions. Described antiviral compound also can be used as solid suspension and uses.

For inhalation, composition is carried by pressurized package or sprayer with the form of aerosol spray usually used according to the present invention, needs to use propellant, such as air, dicholorodifluoromethane, dichlorotetra-fluoroethane or other suitable gas. Preferably antiviral compound preparation of the present invention is mixed in the aerosol propellants, make the as mentioned particle of the inhalable described in the dry powder formulations. This Particles Suspension can be chosen wantonly with the surface-active medicament and be coated with to improve its scattered band in propellant. When using compresed gas aerosol, can provide a valve to carry pre-metering and definite dosage unit.

Can obtain to pass through the commercial blast atomizer that obtains, and can come to carry to object with it the antiviral compound of atomizing. This blast atomizer includes but not limited to: the product that AeroTech 11 (CIS-US, Bedford, Mass.) provides. In addition, for antiviral compound that will atomizing is transported to the lung of object, but additional oxygen originates to provide for example flow velocity of 10L/min on the sprayer. Usually, during spontaneous respiration, the interval that sucks by mouth mask is preferably 5-40 minute. The invention provides new composition, wherein comprise the antiviral compound that suitable carrier and dosage are enough to reduce or alleviate the atomizing of SARS patient's viral load and SARS symptom. This dosage can be lower than to reduce or alleviate SARS patient's viral load and the corresponding whole-body dose of SARS symptom.

Antiviral, SMIP, SMIS of the present invention and immune regulation composite can be treated SARS and SARS symptom with the sterols anti-inflammatory agent. The example of sterols anti-inflammatory agent of the present invention comprises hydrocortisone, prednisolone dexamethasone, Acetospan, FA, fludrocortisone acetate, betamethasone etc.

The antiviral compound major part of the present composition is atomized into the particle that can conduct drugs to bronchioli terminales and respiratory bronchiole. For antiviral compound effectively is transported to the pulmonary branches tracheae space of respiratory tract with aerocolloidal form, the intermediate means diameter of the aerosol particle of formation must be mainly between 1-5 μ m. Said preparation also must not can cause adverse effect to the function of respiratory tract. Therefore, said preparation must contain the medicine that capacity prepares and avoids simultaneously bad reaction with effective delivering medicament under this condition.

For liquid solution and suspension, can from commercially available sprayer, select sprayer. Available from Medicaid and Pari LCS, the blast atomizer of the Sidestream by name 0 of LC Plus and available from Pari Respiratory Equipment, Richmond, the blast atomizer of the eFlow by name of Virginia are the examples that typically is fit to put into practice sprayer of the present invention. Ultrasonic nebulizer can be made the particle that size is about 1-5 μ m, and for example the UltraAire of the Aerosonic of DeVilbiss and Omron is suitable for.

Advantageously, advantage of the present invention also offers the kit that the consumer is used for treating and/or preventing SARS. This kit comprises: (a) contain at least a compound described in listed in the table 34 and 35 for the treatment of effective dose or the listed United States Patent (USP) of table 1, table 2 and table 35 and the disclosed international patent application and the pharmaceutical composition of pharmaceutically acceptable carrier, carrier or diluent; (b) for the container that holds described pharmaceutical composition; And, randomly; (c) specification that treats and/or prevents the method for SARS with this pharmaceutical composition is described. This kit can be chosen the antiviral compound that comprises multiple treatment SARS wantonly, and wherein, described antiviral compound is selected from 3C-sample protease inhibitors and papain-sample protease inhibitors. In another embodiment, this kit comprises a kind of antiviral compound, and described antiviral compound is the dependent RNA polymerase inhibitor of RNA. When this kit comprised multiple antiviral compound, the antiviral compound in the kit can be chosen wantonly and pharmaceutical composition administering drug combinations of the same race.

Used " kit " of the present invention comprises the container that holds different components, such as the bottle of packing or the tinfoil paper packing of packing. Container can be any conventional shape known in the art or form, made by pharmaceutically acceptable material, for example the sack of carton or cardboard case, glass or plastic bottle or tank, resealable (for example be used for " replenishing " tablet pack into different containers) or blister pack (wherein containing the wrapping position of extruding single dosage according to therapeutic scheme). Used container can be depending on related accurate formulation, and for example conventional cardboard case is not used for filling liquid suspension. It is feasible using together a plurality of containers in the packing of marketing unit's formulation. For example, tablet can be installed in the bottle, and bottle can be contained in the box.

An example of this kit is called blister pack. Blister pack is that packaging industry is known, and is widely used in packing unit pharmaceutical dosage form (tablet, capsule etc.). Blister pack is made of for the foil of transparent plastic material upper cover preferred of relatively hard sheet material and its usually. In packaging process, will form depression in the plastic foil. Recess has the single tablet of wanting packaged or the size and shape of capsule, perhaps can have and a plurality of size and shapes of wanting packaged tablet and/or capsule to conform to. Then, tablet or capsule are placed in corresponding recess, and surperficial with relatively hard sheet material sealed plastic paper tinsel with the plastic foil of the opposite direction that forms depression. Consequently, tablet or capsule are by as required separately sealing or be sealed in together in the depression between plastic foil and the sheet material. The intensity of preferred sheet material should be and can form opening by the sheet material of recess manually being exerted pressure at recess, thereby can shift out tablet or capsule from blister pack. Then can from described opening, take out tablet or capsule.

May need to provide a kind of memory of writing auxiliary, include information and/or the instruction of doctor, pharmacist or object during this memory of writing is auxiliary, for example lean against the other digital form of tablet or capsule, these numerals are corresponding with the medication fate that regulation should be taken tablet or capsule, or comprise the card form of same type information. Other auxiliary example of this memory has the schedule that is imprinted on the card, and for example " the first week, Monday, Tuesday " ..., " second week, Monday, Tuesday ... " etc.. Be not difficult to understand other variation that memory is auxiliary. " daily dose " can be tablet or capsule or several tablets or the capsule of specifying that the sky will take. Simultaneously, the daily dose of one or more components of kit can be made of single tablet or capsule, and the daily dose of one or more other components of kit can be made of several tablets or capsule.

Other specific embodiments of kit be design be used for sometime according to they with the distributor of sequentially distributing daily dose. It is auxiliary that the preferred allocation device is equipped with memory, so that observe therapeutic scheme. The auxiliary example of this memory is mechanical counter, and it can represent the daily dose number that has been assigned with. Another auxiliary example of this memory is that the battery-driven microchip stores device that liquid crystal display maybe can be heard cue is housed, and for example, can show the date of taking for the last time daily dose and/or point out the next time Time of Administration of dosage.

Embodiment

The example of embodiment 1-SARS viral isolates

From a patient clinical sample of Frankfurt, Germany, isolate a kind of SARS virus (FRA). Separator is grown in the Vero cell. Extract the RNA of SARS virus and increase by RT-PCR. Virus genomic nucleotide sequence is determined by direct Sequencing PCR product. Computer Analysis is used for predicted gene stack features, relatively this genome and the coronavirus of previously known and the sequence of different SARS virus separators.

Separate and more specific following the carrying out of order-checking. After SARS virus is gone down to posterity for the 3rd time in the Vero cell, by ultracentrifugation from 3 * 10⁷Purified virus particle in the cell conditioned medium. Extract viral RNA with Triazol method (Gibco-BRL). Viral RNA (200ng) is transcribed into cDNA with the thermally-stabilised reverse transcriptase of fowl RNaseH-, transcribes and follows manufacturer's explanation (ThermoScript RT System, Invitrogen). Briefly, use 50pmol few (dT)₂₀(SEQ ID NO:7389) or 25ng random hexamer cause the RT reaction in 20 μ l final volume. Amplification and order-checking SARS genome are finished by direct Sequencing PCR product, obtain the PCR product and use: i) from the special primer of homologue conserved region, homologue is found by the known coronavirus of multiple contrast; Ii) oligonucleotides of design around the short sequence of SARS separator, sequence can obtain on the net by WHO network laboratories; Iii) for the degenerate primer of amplification cDNA mixture, primer and end-product have a plurality of overlapping fragmentses. It is by using the long range PCR (Expand High Fidelity system, Roche) of high-fidelity Taq, using the primer that designs in institute's selected episode that identification Gap closes. Move (primer walking) by the primer step and collect sequence, use BigDye terminator chemistry (Applied Biosystems) and automated DNA sequenator (3700 capillary patterns, Applied Biosystems). Behind first critical point that obtains the complete genome group, one group of forward and reverse primer are used for from the beginning amplification and sequenced genes group, use the template DNA section of average 2kb. AutoAssembler (Applied Biosystems) compiles from the reading of overlapping fragments and manual editing continuous 29 automatically, 740bp.

The Computer Analysis sequence is following carries out. Complete GCG Wisconsin software kit (version 10.0) is used for Computer Analysis gene and protein sequence. PSORT program (http://psort.nibb.ac.jp/) is used for local prediction. For secondary structure analysis, use PHD software, PHD software can obtain http://cubic.bioc.columbia.edu/predictprotein/ in following network address. The PSI-BLAST algorithm is used for homology search (http://www.ncbi.nlm.nih.gov/blast), uses the nonredundancy albumen database. ClustalW is used for obtaining the multiple sequence contrast of gene and protein sequence. The LearnCoil-VMF program is used for the coiled coil district (http://learncoil-vmf.lcs.mit.edu/cgi-bin/vmf) of prediction spike protein. Leucine zipper predicts that with program 2ZIP this program can obtain at http: // 2Zip.molgen.mpg.de..

Carry out system and analyze, the contiguous join algorithm (Felsenstein J 1993 is by the program of author's distribution) of using system's generation inference software kit (Phylip) Program NEIGHBOR to carry out. Bootstrap analyzes usually and carries out service routine Seqboot with 100 kinds of repetition things. Process dendrogram and show with TreeView. Program HMMER is used for producing sequence from the multiple sequence contrast of spike protein S1 domain and distributes. Subsequently, the HMMPFAM program is used for relatively SARS furcella S1 domain and these distributions.

The length of SARS virus isolate gene group is the structure that 29,740 bases and genome general structure are similar to 3 known coronavirus groups. From the terminal beginning of targeting sequencing 5 ', can identify non-translational region (UTR) and 2 overlapping open read frames of a kind of polyprotein of encoding, polyprotein contains and copies essential enzyme. Furcella (S), coating (E), matrix (M), nucleocapsid (N) structural proteins and other 8 ORFs that are specific to SARS virus after them. At genomic 3 '-end, isolate the UTR of poly (A). Lower with total homology of coronavirus group 1,2 and 3, so SARS virus belongs to new coronavirus group (group 4). More detailed spike protein amino acid sequence analysis demonstration SARS virus separator and coronavirus group 2 are more closely related.

The complete genome group length of SARS virus separator is 29,740bp. This sequence can obtain and GC content be 40.8% at Genbank, can compare with the GC content of the known viruse of identical family. Genome structure and other coronavirus structure are similar. Prediction has 14 open read frames. The primary structure of genome and gene outcome is shown in Figure 17 and table 10. 1,2 and 3 of SARS genome and coronavirus group relatively be reported in Figure 18.

Nucleotides 1-73 comprises the RNA targeting sequencing of prediction, is the non-translational region (UTR) that contains 197 nucleotides after the targeting sequencing. Be 2 overlapping open read frames (ORF1a, ORF1b) after the UTR, they contain 2/3rds genome (nucleotides 265-21485). Their large polyproteins of encoding estimate that this polyprotein processes to produce the replicase complex by virus protease. Genomic 3 ' part comprises gene and 8 prediction ORFs (Figure 17) with unknown function of 4 structural proteins of coding (S, spike protein, E, envelope protein, M, matrix glycoprotein and N, nucleocapsid protein). At last, at genomic 3 ' end, we find the 2nd UTR with 340 bases, and its back is poly (A) section. We have identified (IG) sequence between a gene of inferring, and are also referred to as to transcribe correlated series (TAS), and this is the characteristic feature of coronavirus. The feature of IG sequence is to have 6-18 nucleotides and can find in the front of each gene at targeting sequencing 3 ' end. The IG sequence copies middle performance key effect at rna transcription and its. The IG sequence signature of SARS virus is sequence SEQ ID NO:7293 and has 9 times (Figure 17) in genome. Targeting sequencing and IG sequence are special for each coronavirus, represent the special signal of virus.

The replicase zone

Rdrp gene ORF1ab (SEQ ID NO:7232) is that ORF1a and ORF1b form by 2 overlapping ORFs, and this gene can be translated into a single polyprotein, and this is to move the frame ribosomes by the position 13,393 in the polymerase code area. Referring to Brierley etc., Embo J 1987:6 (12): 3779-3785. As expected, there is a stem ring sequence (SEQ ID NO:7390 in 10 base-pair places in this downstream, position; 5 '-CGGTGTAAGTGCAGCCCGT CTTACACCG-3 '). This polyprotein cuts into a plurality of albumen after translating altogether cutting or translation by the protease of himself encoding. Use is cut consensus sequence and is simulated with other coronavirus, we have drawn the collection of illustrative plates of this polyprotein possibility cleavage site and have identified 14 kinds of products, comprise leader protein p28, MHV p65 albumen homology thing and other the 12 kinds albumen (nsp, non-structural protein) (Figure 17 and table 10) of naming nsp13 from nsp1. There are some function motifs in the amino acid sequence analysis prompting in the ORF1ab albumen of inferring. We are specific to have made the collection of illustrative plates of 2 kinds of potential protease (nsp1 and nsp2), a kind growth factor motif in the ORF1a, and in ORF1b, we have identified RNA polymerase (nsp9) and the unwindase (nsp10) of predicting. The cleaved products of other prediction (nsp3, nsp4, nsp5, nsp6, nsp11, nsp12 and nsp13) is the albumen of Unknown Function. Many these albumen infer and are present in the rna replicon complex, and this complex is relevant with membrane structure in the infected cell. The specific hydrophobic domains that comprises of nsp3 and nsp4. As shown in figure 18, SARS replicase zone has with coronavirus group 1,2 and 3 and similarly forms; Yet, overall amino acid conservative low (table 11). Most of conservative protein is polymerase and unwindase.

Nsp1 is class papain cysteine proteinase (PLP), initial 2 protein products (targeting sequencing albumen p28 and p65 homologue) of its cutting. In the nsp1 of MHV, (Kanjanahaluethai etc. (2000) J.Virol 74 (17): 7911-21), these domains are also guarded in ox infectiousness enterogastritis virus (TGV) and people 229E coronavirus to have made 2 collection of illustrative plates that the domain of class papain activity arranged. Yet by contrasting with SARS nsp1 sequence, we have identified a unique PLP domain that contains catalytic residue Cys833 and His994.

Nsp2 is chymotrypsin-picornavirus class HRV 3CP (3CLp), process after the translation of responsible other 12 kinds of albumen, most of these albumen are at Q/A or Q/S site cutting (Ziebuhr etc. (1999) J.virol 73 (1): 177-85). Nsp2 also has the autoproteolytic cleavage activity. Main catalytic residue is conservative and be positioned at position His41 and Cys145 preferably in other coronavirus. In addition, in SARS 3CLp sequence even found conserved amino acid Tyr161 and His163, they are considered to participate in substrate identification and for essential (Hegyi etc. (2002) J.Gen Virol 83 (Pt3): 581-593) of proteolytic activity.

Invention comprises the 1ab sequence of SEQ ID NO:9960 and the orf1a sequence of SEQ ID NO:9961, comprises fragment, variant, homologue etc.

Structural region

The nucleotide sequence of analyzing SARS genome 3 ' part has identified 12 prediction open read frames. They are encoded in 8.2kb and comprise 4 kinds of structural proteins S, E, M and N, and these albumen are general for all coronavirus and 8 prediction ORFs, and they are specific to this virus. The special IG sequence of SARS-(Tu17 ﹠18) the prompting most gene of most of ORFs upstream may independently be transcribed. What is interesting is that the sequence identical with group 2IG also is present in the front of RNA targeting sequencing end and matrix encoding gene and ORF.

Furcella is I type glycoprotein, and it forms large furcella and responsible receptors bind and film in virosomal surface and merges (Gallagher (2001) Adv Exp Med Biol 494:183-92). Long 1255 residues of this albumen have 17 prediction N-glycosylation sites. It has 13aa leader peptide and the terminal film anchor series (1202-1218) of 17aa C-. Some (MHV, HCoV-OC43, AIBV and BCoV) but not all (TGV, FIPV, HCoV-229E) coronavirus spike protein be the proteolysis cutting in 2 subunit S1 and S2. Suppose that S1 forms ball heads, ball heads keeps terminal film non-covalent connection of anchor with C-. Cutting is regulated by the basic amino acid sequence, and the consensus sequence of the similar furin cleavage site of this sequence (Garten etc., Biochimie 1994; 76 (3-4): 217-225). Yet in the situation of this SARS virus separator, we fail to identify this sequence, show that the S albumen of this SARS virus separator unlikely cuts in maturation. The spherical structure of secondary structure prediction indication spike protein is conservative in all known coronavirus. The S1 domain is mainly formed by the β lamella and may adopt spherical folding, and in the S2 domain, prediction has widely alpha helical region. In addition, in the LearnCoil-VMF program prediction S2 of particular design for the identification of the similar district of coiled coil in the fusion albumen 2 coiled coils are arranged, respectively across amino acid 900-1005 and 1151-1185 (Figure 19). 2 coiled coil districts all comprise the leucine zipper motif, and this motif also exists in the furcella of all coronavirus. Known leucine zipper promotes the albumen oligomerization; Because the spike protein of TGV and MHV forms heterotrimer (Delmas etc., J Virol1990; 64 (11): 5367-5375) (Godeke etc., J Virology 2000; 74 (3): 1566-1571), can imagine it in the SARS leucine zipper, play promote and/or stable phase like the effect of quaternary structure. Spike protein is brought into play Main Function in coronavirus biology, because the S1 domain comprises in receptors bind domain and the virus and epitope, and the S2 domain participates in the film fusion process essential to viral infection. As expected, the contrast of the multiple sequence of different spike proteins shows that the variability degree in the S1 domain is large, and S2 is more conservative.

Envelope protein E is the polypeptide of lacking very much, and 76 amino acid are arranged, and the form generation of participation virion coating (Godet etc., Virology 1992; 188 (2): 666-675). The Computer Analysis prediction has long span membrane structure territory and 2 N glycosylation sites of close N end. Very low with the amino acid similarity of other coronavirus, maximum homology is the little envelope protein with infectiousness enterogastritis virus (TGV).

Matrix glycoprotein (M) is the polypeptide that 221 residues are arranged, and predicted molecular weight is 25kDa. The topological structure of Computer Analysis prediction is comprised of short amino terminal extracellular domain, 3 TMDs and the carboxyl terminal that is positioned at the peplos inboard. With the matrix glycoprotein simulation of TGV, avian infectious enterogastritis virus (AIBV) and super virulence MHV-2 bacterial strain, the terminal N-glycosylation of the comfortable N of SARS M sugar egg. SARS M glycoprotein shows and group 2 viral similitudes the highest (table 11).

At last, nucleocapsid protein N is the phosphoprotein of long 397 residues, interacts to form nucleocapsid with virus genome RNA. Low with the conservative level of other coronavirus, scope from 26.9% homogeneity of HCoV-229E to 37.4% homogeneity (table 11) of bovine coronavirus (BcoV). The Characterization of antigenic epitopes of nucleocapsid protein finish (Li etc. (2003) Geno Prot﹠Bioinfo 1:198-206), wherein the epitope position of PROTEIN C end is positioned SEQ ID NO:7394 (the amino acid 371-407 of SEQ ID NO:6052).

Except top basic albumen, one group of other peptide of many expressing virals, these peptides are generally unessential for viablity, but can affect the Viral infection potentiality (de Haan et al., Virology 2002; 296 (1): 177-189). These albumen are generally conservative in the member of identical serum group, but remarkable at group difference. For this reason, they generally are called group differential protein (Figure 11). Group 1 member here by HcoV-229E as representative, have 2 group specific genes, be positioned between S and E gene and sometimes in N gene downstream 1 or 2 ORFs, before genomic 3 ' UTR district. Group 2 virus with MHV as prototype, have 2 between ORF1b and S group specific gene (2a and HE) and other 2 at S and intergenic group of specific gene of E. At last, organize 3 viruses by prototype AIBV as representative, have intergenic 2 the group specific genes of S and E and other 2 at M and intergenic group of specific gene of N.

Except the hemagglutinin esterase HE that proves blood clotting and acetylesterase enzymatic activity, all organize special ORFs encoding proteins, and the effect of albumen is not yet definite.

What is interesting is that specific gene is arranged ORFs unique and prediction and do not shown with the existing ORFs of other coronavirus any significant homology is arranged in the SARS genome, also from any other known albumen from different organisms remarkable homology is not arranged. The virus of picture group 1 and 3, SARS lacks the HE hemagglutinin and is not included in ORF1b and the intergenic ORFs of S. In addition, encode in 2 kinds of prediction ORFs (ORF3 and ORF4) zones between S and E, superposition is in they most length. ORF3 has the IG sequence, at ATG upstream from start codon 2bp. Opposite with other group, SARS is at M and 5 predictions of the intergenic district inclusion of N ORFs. ORF7 is positioned at 10 base places, M gene terminator codon downstream, and the IG sequence that has is at 155 nucleotides of ATG upstream from start codon. Similarly, the IG that presents of ORF8 and ORF10 is just at the ATG upstream from start codon. On the other hand, the 5 ' end of ORF9 and ORF11 briefly superposes with flanking gene, and for this reason, they do not need the IG activated transcription. ORF12 and N gene superpose fully and are very low with the 22kDa albumen homology of MHV virus, and the latter encodes in the corresponding region.

Although do not have the indication of possibility location and obtain function from the sequence similitude, ORF3, ORF7 and ORF8 comprise hydrophobic section, point out relevant with membrane structure. In addition, the longest ORF3 is the ORF (table 11) that unique coding contains the peptide of a large amount of prediction O-glycosylation sites in the SARS specific gene. In ORF3, ORF11 and ORF12, identify the N-glycosylation site of prediction.

2 shorter ORFs in the non-structural area are SEQ ID NOS:9965 and 9966. Invention comprises the polypeptide with these sequences, and fragment, variant etc.

System analyzes

Use in the past from the replacement frequency in 922 conservative bases of 11 kinds of coronavirus pol genes of 3 different serum group and show that the variability among each serum group member is little more many than the variability between different serum group, confirm the grouping of above-mentioned serology (Stephensen etc., Virus Res 1999; 60 (2): 181-9). We use the 922bp zone of SARS pol gene and it and same clip from other 12 kinds of coronavirus are compared. The gained dendrogram shows that SARS virus is different from other 3 coronavirus groups (Figure 20). Use obtains similar results (data are not shown) from the full length amino acid sequence of pol, 3CL-protease and the unwindase in replicase zone and from the furcella of structural region and the full length amino acid sequence of matrix glycoprotein. The complete genome group of the SARS virus of these data validations in a new coronavirus group (group 4) bunch.

Be the possible evolutionary relationship of clearer understanding, we use the consensus sequence in albumen predict territory to analyze. We are specific to have produced from the consensus sequence of group 1 with the spike protein S1 domain of group 2, and we compare them with the S1 domain of SARS furcella subsequently. Fail to produce consensuses from organizing 3, because the spike protein of known AIBV only. What is interesting is that the tree that makes up by contrast SARS S1 and the consensus that produces from 2 groups of spike proteins is different from the tree among Figure 20, the SARS that its shows organizes 2 in close relations many (Figure 21 A) with coronavirus. Further analyze and show that 19 in existing 20 cysteines are spatially conservative with group 2 consensus sequences in the SARS S1 domain, and only 5 keep in group 1 and group 3 sequences (Figure 21 B). Consider the basic role that cysteine is brought into play in protein folding, possible SARS S1 domain and coronavirus group 2 are shared a similar space structure.

Sequence variability between sars coronavirus

We compare FRA sequence and online obtainable 4 kinds of complete SARS genomes. Detect altogether 30 sudden changes. 9 silences in these sudden changes, and 21 cause 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (table 12). In ORF1a, detect 3 silences and 7 productivity sudden changes (productive mutations). In ORF1b, 5 silences and 3 productivity sudden changes are arranged. A kind of productivity sudden change is caused that by 2 nucleotides replacements 2 nucleotides replace and cause the single amino acids variation. 5 kinds of variations are positioned in the spike protein, and wherein 4 is that productivity and 1 are reticent. 2 productivity sudden changes are arranged among ORF3 and the matrix glycoprotein M. 1 productivity sudden change is arranged among ORF10 and the nucleocapsid protein N.

Total variances between FRA and TOR2 is 9 nucleotides, produces 2 silent mutations and 7 amino acid variations. Total variances between FRA and Urbani is 12 nucleotides, produces 5 silent mutations and 7 amino acid variations. For CUHK, 16 nucleotides differences are arranged, wherein 5 is silent mutation. For FRA and HKU 14, nucleotides changes 4 silences of generation and 9 productivitys sudden changes.

Embodiment 2-production, deactivation and purifying intact SARS virus are used MCS chromatographic resin purifying, then use density gradient ultracentrifugation

SARS virus separator FRA1 (EMBL:AY310120) goes down to posterity at the VERO cell, cell is incubated at DMEM (Gibco: catalog number (Cat.No.) 21969-035, lot number 3078864), penicillin/streptomycin (Gibco: catalog number (Cat.No.) 15070-063, lot number 1120042) and 3%FCS (Gibco: catalog number (Cat.No.) 10270-106, lot number 40F6130K), at 37 ℃, 5%CO₂ Use trypsase (Gibco: catalog number (Cat.No.) 25300-054, lot number 3078729) so that cell detachment.

For generating virus, go down to posterity for inoculation VERO cell for the 3rd time, moi is ~ 0.1. Cell was hatched 1 hour with 37 ℃ in virus in infectious culture medium (without DMEM, the FCS of PS); After 1 hour, cell wash 2 times and when 3%FCS and antibiotic exist 37 ℃ hatched again 48 hours. (p.i.) collected supernatant in 48 hours after infecting, by coming pre cleaning in centrifugal 10 minutes 4 ℃ of 3000rpm.

Deactivation of SARS virus be by β-propionic acid lactone (BPL) 4 ℃ of processing (1: 2000) 18 hours, then 37 ℃ 3 hours. Whether successful deactivation of Test Virus, the VERO cell was hatched 4 days at 37 ℃ with the supernatant that 10ml BPL processes; Subsequently, supernatant is transferred in the fresh VERO cell culture and was hatched 4 days again. Check the CPE (CPE) of cell.

The SARS virus gleanings of 200ml BPL-deactivation is used the clarification of 0.65 μ m-hole dimension filter (47mm diameter) subsequently, so that virion is passed through and the maintenance cell fragment. Filter unit connects the Masterflex pump, and this pump is realized 40ml/ minute constant flow rate.

A.MCS chromatographic purifying step

The viral suspension that filters carries out the MCS chromatography subsequently. The following preparation of MCS post. The 27ml slurries produce 14ml deposition resin, and resin is filled with G_tec Superformance post (diameter 1.0cm, high 15.7cm, volume 12.33ml). 1% acetone soln of 1% column volume injects post and post runs with 100cm/ hour flow velocity. Subsequently, HETP, N and A_sValue be can be regarded as HETP:0.056cm, N/m:1790 and A_s＝1.20。

Protein content after the assessment MCS chromatographic step in the purification solution is with two cinchonic acids (BCA) method ((Interchim) (referring to for example http:www.piercenet.com/files/bca.pdf) and electrophoresis.

SDS-PAGE is according to Laemmli, and Nature (1970) 227:680-685 carries out. The Sample Dilution of SDS-PAGE is 77 μ g/ml to protein concentration. Add different protein concentrations, this depend on used gel kind (10/12/15 hole, Novex/Invitrogen):

Hole count	The albumen diluted concentration	Application of sample	Albumen/hole
				10 holes	77μg/ml	20μl	1μg
12 holes	77μg/ml	15-20μl	0.75-1μg
				15 holes	77μg/ml	10μl	0.5μg

Be used for the following preparation of sample of reproducibility SDS-PAGE:

	26 μ l sample or dilute samples
		+ 10 μ l NuPage sample buffer (4x) SDS NP0003
	+ 4 μ l TCEP Bondbreaker solution 77720 (in MilliQ water 1: 2)
		Final volume:	40μl

Sample keeps 1 hour (sample stay M albumen that room temperature can prevent coronavirus condense/form complex) in 70 ℃ of heating 10 minutes or room temperature, subsequently in desk centrifuge with 14,000 centrifugal about 1 minute.

Be used for the following preparation of mark of gel. Contain the gel band that is less than 1 μ g albumen and easily present by the silver process of dying, use silver-colored transfection reagent box protein to add a kind of dying operation (Pharmacia Biotech).

Western blotting is following to carry out. Half-dried engram technology is used for albumen is transferred to nitrocellulose membrane from sds gel. Transfer 0.8mA/cm²Electric current carried out 1 hour. The rabbit polyclonal antibody of anti-SARS virus is used for carrying out immunity to be surveyed, use Western Breeze, the Novex Western blotting immunity detection reagent (Novex/Invitrogen) that develops the color.

The tomographic map that deactivation SARS MCS catches step is shown in Figure 27. For estimating purity, by dying to analyze MCS chromatography part at NuPage 10% or the upper silver of 4-12% Bis-Tris-gel (Novex), it is under reducing condition that silver dyes, 10 minutes (Figure 28) of 70 ℃ of heating. These parts are also passed through western blot analysis (Figure 29) under the same conditions to estimate purity, use PAK 11/03 SARS Cov 270603, and neutralization is tired 1: 512 (this antibody is used for specifically and Western blotting subsequently). Purity is estimated as follows:

Sample	Volume/ml	[albumen]/μ g/ml	Total protein/mg	Step albumen recovery/%
					The coronavirus gleanings	100	2547.6	254.76	100
After filtering=application of sample	100	2440.3	244.03	95.8
					Flow through	85	2321.4	197.32	77.5
Wash	49.32	468.5	23.11	9.1
					Peak 1	12.12	252.7	3.062	1.2
The total recovery	-	-	464.4	86.5

B. density gradient ultracentrifugation step

The SARS virus part of wash-out is carried out density gradient ultracentrifugation subsequently, uses swinging bucket rotor to be further purified inactivation of viruses. 3ml MCS peak part application of sample (15-60% sucrose on linear gradient; 17ml 15% and 17ml 60% sucrose are in gradient mixer). Separate with Beckman SW 28 rotary heads and carried out 2 hours with 20,000rpm.

Chart and the purity among Figure 31 that sucrose in the linear gradient ultracentrifugation part and protein content are shown among following table, Figure 30 are estimated:

Part	Part size/ml	[sucrose]/%	[albumen]/μ g/ml
				1	2	61	96.12
2	2	59.4	98.62
				3	2	57.5	87.63
4	2	54.5	86.91
				5	2	50.5	79.9
6	2	47.2	74.3
				7	2	43.7	68.05
8	2	40.2	60.43
				9	2	37.2	57.38
10	2	34	53.12
				11	2	30	50.63
12	2	25.7	35.02
				13	2	22.4	35.33
14	2	19.5	39.25
				15	2	15.5	69.79
16	2	8.5	169.03
				17	2	8.5	128.96

Relative standard's curve is the protein concentration of measure portion 11 (Figure 31 SDS-gel) again, and calibration curve prepares in 30% sucrose and makes protein concentration is 3.67 μ g/ml (0.05 μ g on the gel). As if M albumen lacks in these goods, and this may be to cause (heated sample) by the sample treatment process.

Because the sucrose of this mensuration disturbs, the protein concentration of table 2 is measured may be variant.

Embodiment 3-production, deactivation and purifying intact SARS virus are used MCS chromatographic resin purifying, then use density gradient ultracentrifugation

Deactivation of SARS virus is such as preparation as described in the top embodiment.

A.MCS chromatographic purifying step

In this example, 200ml deactivation of SARS virus gleanings carries out the MCS chromatography. The tomographic map that the deactivation SARS viral purification of use MCS is caught step is shown in the albumen recovery of Figure 32, following table and the purity estimation of Figure 33.

Sample	Volume/ml	[albumen]/μ g/ml	Total protein/mg	Step albumen recovery/%
					The coronavirus gleanings	200	2239.2	447.83	100
After filtering=application of sample	200	2245.1	449.02	100.3
					Flow through	185	2126.3	393.37	87.8
Wash	49.32	450.1	22.2	5.0
					Peak 1	4.43	1245.6	5.52	1.2
The total recovery	-		421.08	93.7

B. density gradient ultracentrifugation step

3.5ml MCS peak part is loaded onto (15-40% sucrose on the linear gradient subsequently; 16ml 15% and 16ml 40% sucrose are in gradient mixer). Separate with Beckman SW 28 rotary heads and carried out 2 hours with 20,000rpm.

Sucrose in the linear gradient ultracentrifugation part and protein content are shown in the chart among following table and Figure 34:

Pipe	Part size/ml	[sucrose]/%	[albumen]/μ g/ml
				1	2	40	45.86
2	2	39	45.68
				3	2	37.5	44.14
4	2	35.5	37.82
				5	2	33.5	34.48
6	2	31.5	31.76
				7	2	30.5	29.49
8	2	28	30.87
				9	2	25.5	31.7
10	2	23.5	26.74
				11	2	21.75	23.58
12	2	20	35.33
				13	2	18	96.38
14	2	14.5	523.79
				15	2	8	941.97
16	2	8	696.7

The albumen recovery is shown in following table and purity estimates to be shown in Figure 35. Density gradient part 8,9 and 10 electron micrograph are shown in Figure 36:

Step	Volume/ml	Albumen/μ g/ml	Total protein/mg	Step albumen %
					Application of sample	3.5ml	1245.6	4359.6	100
Large quantities of protein parts	3.5ml	720.8	4324.9	99.2
					Virus honeybee part	8ml	29.7	237.6	5.5
The total recovery			4562.5	104.7

Embodiment 4-deactivation of SARS virus immune mouse

Mouse at the 0th, 14 and 28 day with 5 μ g BPL-deactivation SARS-CoV particle (BPL-SARS-CoV) subcutaneous inoculations, separately or with Alum or MF59 as adjuvant. At the 0th (before the immunity), 13 (after the 1st immunity), 28 (after the 2nd times) and 35 days (1 week after the 3rd immunity) collection serum. The assessment neutralizing antibody is at extracorporeal blocking SARS-CoV vero cells infection. After 3 immunity, neutralization is tired scope 1: 100-1: 1000, and level is similar to the level that exists in SARS convalescent's serum. As shown in the table, after the 3rd immunity, induce neutralizing antibody without Adjuvanted vaccines, can significantly improve this vaccine potency by comprising adjuvant, neutralizing antibody occurs and total titer increase after the 3rd immunity thereupon afterwards the 2nd immunity.

Immunogene	Neutralization is tired
					Before	After the 1st time	After the 2nd time	After the 3rd time
	BPL-SARS-CoV+MF59(5μg)	＜1∶20	＜1∶20	1∶158					1∶630
BPL-SARS-CoV+Alum(5μg)					＜1∶20	＜1∶20	1∶67	1∶612
	BPL-SARS-CoV(5μg)	＜1∶20	＜1∶20	＜1∶20					1∶71
PBS					＜1∶20	＜1∶20	＜1∶20	＜1∶20

Embodiment 5-uses deactivation of SARS virus immunity Balb/c mouse

Developed the Balb/c mouse model that is used for SARS and infects (Subbarao etc. (2004), J.Virol., 78:3572-77). In this model, the Balb/c mouse is with 10⁴TCID ₅₀The virus intranasal vaccination. Inoculate rear 48 hours, in the infecting mouse lung, can detect TCID₅₀Virus titer increases 2-log. Although virus replication easily detects, mouse does not show any SARS disease symptoms and 1 all spontaneous removing virus after inoculation. The virus titer of the animal of previous immunity is compared control-animal and is reduced, and proves the protection effect of the vaccine of assessing.

In this example, every group of 4 Balb/c mouse are with 3 times (the 0th, 14,28 day) of 5 μ g BPL-deactivation SARS-CoV particles immunity, separately or in conjunction with MF59, at the 43rd day with 10⁴TCID ₅₀SARS-CoV attacks. After the virus attack 2 days, mouse euthanasia, quantitatively the SARS-CoV of concha (NT) and lung also measures the average virus titer of each mouse. Control group is accepted independent PBS, or accepts influenza virus (FLU), is with or without the MF59 adjuvant. Data following (also referring to Figure 51), wherein 4 mouse of every group of test and virus titer are expressed as log₁₀TCD ₅₀Every gram tissue:

Immunogene	The virus replication in the mouse lung under fire		The virus replication in the mouse concha under fire
					# infection/# test	Mean value (± SE) virus titer	# infection/# test	Mean value (± SE) virus titer
	PBS	4/4	6.3±0.3	3/4					2.8±0.35
Independent MF-59					4/4	6.1±0.13	3/4	3.0±0.38
	FLU vaccine (5 μ g)	4/4	6.3±0.07	3/4					2.9±0.36
FLU vaccine (5 μ g)+MF-59					4/4	6.0±0.19	4/4	3.0±0.11
	BPL-SARS-CoV(5μg)	_	1.6±0.13 *	0/4					Do not detect**
BPL-SARS-CoV(5μg)+   MF-59					0/4	Do not detect*	0/4	Do not detect**

Compare with the PBS immune mouse, two tail Si Shi t checks show: * P＜0.00001 or * * P=0.025

As shown, can not detect virus in the BPL-SARS-CoV immune mouse. The lower limit that infectious virus detects in the 10%w/v LH suspension is 1.5log₁₀TCID ₅₀/ gm, boundary is 1.8 log in the 5%w/v concha suspension₁₀TCID ₅₀/ gm. Therefore, the virus titer in the immune mammal is lower than these initial values.

Therefore, deactivation SARS-CoV vaccine is very effective in pre-preventing virus infection, with in 8 mouse of vaccine only 1 infected, vaccine is with or without the MF59 adjuvant. Do not observe similar protectiveness in control group, control group is PBS dilution, MF59 adjuvant or influenza virus vaccine, and vaccine is with or without the MF59 adjuvant.

After the 1st immunity after 2 weeks, the 2nd immunity after 1 week and the 3rd immunity 1 week assessment take from the Attack Research neutralization of animal blood serum and tire. The mouse of the vaccine immunity of apparatus MF59 adjuvant has developed 1: 71 neutralization and has tired after the 2nd immunity, after the 3rd immunity, be increased to 1: 588, and the mouse of accepting without Adjuvanted vaccines is 1: 64 tiring without any the neutralization after neutralization activity and the 3rd immunity after the 2nd immunity. The serum of each control group mice does not show that any neutralization is active. These data are the ability of the vaccine-induced protectiveness SARS neutralizing antibody of clear proof deactivation SARS-CoV not only, also proves with adjuvant and makes the beneficial effect that vaccine is tired to improving neutralization.

Embodiment 6-preparation contains the OMV of SARS virus antigen

Escherichia coli (E.coli) are with plasmid interested (encoding SARS viral antigen) transfection. Single bacterium colony 37 ℃ of overnight growth in 20ml LB/Amp (100 μ g/ml) liquid culture that plasmid interested is arranged. Bacterium in the 1.0L fresh culture 1: 30 the dilution and 30 ℃ or 37 ℃ of growths, until OD₅₅₀Reach 0.6-0.8. IPTG abduction delivering recombinant protein with final concentration 1.0 mM. After hatching 3 hours, by collecting bacterium and be resuspended in 20ml 20mM Tris-HCl (pH 7.5) and intact proteins enzyme inhibitor (Boehringer-Mannheim in centrifugal 15 minutes 4 ℃ of 8000 * g^TM). All processes subsequently are all at 4 ℃ or carry out on ice.

Cell breaks by ultrasonic degradation, uses Branson Sonifier 450, and with centrifugal 20 minutes of 5000 * g with the not damaged cell of precipitation and inclusion body. The supernatant that contains film and cell fragment was with centrifugal 75 minutes of 50000g (Beckman Ti50,29000rpm), and with 20mM Bis-tris propane (pH 6.5), 1.0M NaCl, 10% (v/v) glycerine is washed, again with 75 minutes precipitations of 50000g. Precipitation is resuspended in 20mM Tris-HCl (pH 7.5), 2.0% (v/v) flesh aminoacyl, intact proteins enzyme inhibitor (1.0mM EDTA, final concentration) and hatches 20 minutes with the dissolving inner membrance. Cell fragment precipitated in centrifugal 10 minutes by 5000g and supernatant with centrifugal 75 minutes of 75000g (Beckman Ti50,33000rpm). Adventitia is washed with 20mM Tris-HCl (pH 7.5), with centrifugal 75 minutes of 75000g or spend the night. OMV finally is resuspended in 500 μ l 20mM Tris-HCl (pH 7.5), 10%v/v glycerine. Protein concentration is measured (Bio-Rad) estimation by standard Bradford, and the protein concentration of interior membrane portions is determined with DC protein determination (Bio-Rad). Different piece from separation process is measured with SDS-PAGE.

Immunogenicity, dosage and the approach arrangement of embodiment 7-restructuring spike protein in mouse

Immunogenicity, approach and the dosage of restructuring spike protein in mouse of assessment invention uses following detailed operation. The antigen of using preferably causes neutralizing antibody at least in the 1/100-1/1000 scope. The energy test increases the antigen of dosage, scope from 5 to 20 μ g restructuring furcella antigen, and independent or mixing equal-volume MF59-citrate is used SC or IM to anesthetized mice in 100 μ l inoculums. The BALB/c mouse group is strengthened the 0th day sensitization and at the 14th and 28 day, processes 6 at every turn.

Group	Process	Dosage/approach	Sampling interval	1	Number of mice
						1-3	The Rec- spike protein	20，10，5μg/ SC	7,21,35,42 days	6 every dosage levels
4-6	The Rec- spike protein	20，10，5μg/SC	7	6 every dosage levels
					7-9	The Rec- spike protein	20，10，5μg/ IM	7,21,35,42 days	6 every dosage levels
10-12	The Rec- spike protein	20，10，5μg/IM	7	6 every dosage levels
					13-15	Rec-furcella-MF59	20，10，5μg/ SC	7,21,35,42 days	6 every dosage levels
16-18	Rec-furcella-MF59	20，10，5μg/SC	7	6 every dosage levels
					19-21	Rec-furcella- MF59	20，10，5μg/ IM	7,21,35,42 days	6 every dosage levels
22-24	Rec-furcella- MF59	20，10，5μg/IM	7	6 every dosage levels
					25	MF59	NA/ SC	7,21,35,42 days	6+6 (killing in the 7th and 42 day)
27	MF59	NA/ IM	7,21,35,42 days	6+6 (killing in the 7th and 42 day)
					29	Salt solution	NA/SC	7,21,35,42 days	6+6 (killing in the 7th and 42 day)
31	Salt solution	NA/ IM	7,21,35,42 days	6+6 (killing in the 7th and 42 day)

This operation also can distribute for assessment of the Th1/Th2 of the Immunel response that causes of restructuring spike protein. Tire with the furcella specific antibody assessment neutralization in the 7th, 21 and 35 day; IgG2a vs IgG1 isotype at the 21st and 35 day definite furcella specific antibody; Respectively in the external hyperplasia of the 7th and 42 day definite lymph node and the anti-restructuring spike protein of splenic t-cell; IFN-γ and IL-4 at the 42nd day anti-restructuring spike protein of assessment splenic t-cell generate, and the restructuring spike protein is from SARS-CoV. Collected peripheral blood at the 7th, 21 and 35 day; Collected LNC at the 7th day, the 42nd day collection splenocyte. Determine respectively to neutralize to tire and isotype with the furcella specific antibody by SARS-CoV vero cells infection and ELISA. Lymph node and splenic t-cell hyperplasia are passed through³[H]-thymidine absorbs to be determined. The T lymphocyte generates the frequency of spleen IFN-γ and IL-4 and determines by ELISPOT and FACS.

Immunogenicity, dosage and the approach arrangement of embodiment 8-spike protein in rabbit

Immunogenicity, approach and the dosage of restructuring spike protein in rabbit of assessment invention uses following detailed operation. The energy test increases the antigen of dosage, scope from 5 to 40 μ g restructuring furcella antigen, and independent or mixing equal-volume MF59-citrate is used SC or IM to anesthetized animal in 200 μ l inoculums. New Zealand white doe is organized immunity as shown in the table, processes 10 at every turn. Animal is strengthened the 0th day sensitization and at the 14th and 28 day. Collected peripheral blood at the 7th, 21 and 35 day. Determine respectively to neutralize to tire with the furcella specific antibody by suppressing SARS-CoV vero cells infection and ELISA.

Group	Process	Dosage/approach	The sampling interval	Rabbit quantity
					1-4	The total length spike protein	40，20，10，5μg/ SC	7,21,35 days	10 every dosage levels
5-8	The total length spike protein	40，20，10，5μg/ IM	7,21,35 days	10 every dosage levels
					9-12	The spike protein of brachymemma	40，20，10，5μg/ SC	7,21,35 days	10 every dosage levels
13-16	The spike protein of brachymemma	40，20，10，5μg/ IM	7,21,35 days	10 every dosage levels
					17-20	Total length spike protein-MF59	40，20，10，5μg/ SC	7,21,35 days	10 every dosage levels
21-24	Total length spike protein-MF59	40，20，10，5μg/ IM	7,21,35 days	10 every dosage levels
					25-28	Spike protein-the MF59 of brachymemma	40，20，10，5μg/ SC	7,21,35 days	10 every dosage levels
29-32	Spike protein-the MF59 of brachymemma	40，20，10，5μg/ IM	7,21,35 days	10 every dosage levels
					33	MF59	NA/ SC	7,21,35 days	10
34	MF59	NA/ IM	7,21,35 days	10
					35	Saline	NA/ SC	7,21,35 days	10
36	Saline	NA/ IM	7,21,35 days	10

Immunogenicity and the dosage arrangement of embodiment 9-restructuring spike protein in ferret

Immunogenicity and the dosage of restructuring spike protein in ferret of assessment invention uses following detailed operation. 3 groups of ferrets, 6 for the treatment of the restructuring SARS-CoV spike protein immunity from Chinese hamster ovary celI system of, ferret, separately or mix equal-volume MF59-citrate, uses SC to anesthetized animal in 200 μ l inoculums. Restructuring spike protein vaccine is tested when senior middle school and antibody titer in causing mouse, strengthens rear 35 days at the 2nd time. Animal is strengthened the 0th day sensitization and at the 14th and 28 day. Collected peripheral blood at the 7th, 21 and 35 day. Determine respectively to neutralize to tire with the furcella specific antibody by suppressing SARS-CoV vero cells infection and ELISA.

Group	Process	Dosage/approach	The sampling interval	The ferret number
					1&2	The Rec-spike protein	Y μ g or 2Y μ g/ SC	7,21,35 days	6
3&4	Rec-spike protein+MF59	Y μ g or 2Y μ g/ SC	7,21,35 days	6
					5	Salt solution	NA/ SC	7,21,35 days	6

3 groups of ferrets that are used for top immunogenicity research, can protect immune animal to avoid infecting and/or the effect of disease for assessment of the restructuring spike protein subsequently by 6 every group. After strengthening for 2 weeks in the tracheae the last time, with 10⁶Intermediate structure is cultivated the (TCID of infective dose unit₅₀) SARS-CoV Utah bacterial strain attack anesthetized animal. Wipe away to assess SARS-CoV infection, as mentioned above (12) by from animal, got nose, pharynx (faringeal) and rectum cotton in rear 20 days in attack. Measure to assess the existence of SARS-CoV in the sample material by RT-PCR and Vero cell infection. The SARS clinical symptom of monitor animal, this is by assessing the length of one's sleep, temperature, respiratory symptom, diarrhoea, body weight and survival. Determine protectiveness by viral burst size and duration and disease symptoms severity and surviving animals percentage.

Embodiment 10-expresses the spike protein that is used for immunity

The SARS-CoV spike glycoprotein is expressed with total length and clipped form, uses said n Sh and nSh Δ TC pCMVIII to make up, and 2 have six histidine marks. Be transfected into 293 cells and COS7 cell and assessing after 48 hours the expression of vector construction. Only in cell lysate, detect total length spike protein (nSh) by Western blotting, and do not have (Figure 52) in the culture medium.

Most of SARS-CoV total length spike protein is macromolecule glycoprotein at the COS7 of transient transfection cells, runs (Figure 53) with 540kDa on the irreducibility gel. Gp540 is thermal instability, and this is that to be dissociated into monomeric form (gp170 ﹠ gp180) fully when boiling indicated, but its anti-DTT processes. The non-covalent homotrimer (gp540) that is unified into of these Notes of Key Data restructuring spike proteins. Confirm also that in deactivation, purifying SARS-CoV virion there is spike protein in homotrimer in connecting. Western blot analysis virion albumen produces almost identical result, used condition identical with definite spike protein feature (Figure 54).

Embodiment 11: spike protein processing

For determining the feature of spike protein processing, infect the BHK-21 cell with the Alphavirus replicon particle of expressing SARS-CoV total length furcella. After infecting 6 hours with MOI 5, infected cell L-[³⁵S] methionine/cysteine mark 1 hour and following the trail of to 4 hours. [³⁵S] spike protein of mark digests by anti-SARS rabbit anteserum immunoprecipitation and with Endo-H. Digestion and not digestible protein all pass through SDS-PAGE (4% polyacrylamide) analysis. Shown in Figure 55, the total length spike protein synthesizes Endo-H sensitiveness high mannose glycoprotein (gp170, ER form), and its experience modifies to become the Endo-H of complete oligosaccharides resistance glycoprotein (gp180, golgiosome form). Gp170 is transformed into the gp180 form (Figure 56) occured in 2 hours.

Embodiment 12: high-level protein expression

For the system of development for rapid expressing protein antigen, use DNA transfection 293 (human embryo kidney (HEK)) cell, to obtain the recombinant antigen of milligram quantities. Prevailing cultivation and rotaring redyeing 293 cell method are in static state or monolayer cultivation thing. Revise these processes, be used for generating secretion or intracellular protein by the transfectional cell in extensive rotaring redyeing 293 cell in suspension and the expansion suspension culture. Some initial experiment are carried out to determine optimal conditions, such as the ratio of cell number, transfection reagent type (FuGENE 6, Lipitoid or RO-1538) and DNA and transfection reagent at 100 milliliters of scale cultures. On the preliminary experiment basis, FuGENE 6 is best transfection reagents.

The dynamics that icp gene is expressed and the dynamics of other viral envelope glycoprotein, the stable protein expression of the Notes of Key Data reached the highest in 72 to 96 hours after transfection, significantly minimizing reaches the top and depends on interested gene afterwards. Therefore, use optimal conditions, transfection method is to scale from 100ml to 4 liter. 4 liters of cultures can be used for generating rapidly 2-10 milligram proteantigen. For promoting output and the recovery of antigen purification and maximization purifying protein, by optimizing transfection conditions with serum free medium.

Large quantities of transfection methods are used for expressing brachymemma and total length spike protein. The dynamics of expressing the spike protein clipped form is shown in Figure 56 A. The expression of spike protein clipped form reached the highest and stable at about 48 hours until 72 hours, therefore collected culture after transfection in 72 hours.

The concentrated 20X of the culture medium of collecting also is used for the spike protein of purifying brachymemma, and purifying is by a very simple purification strategy, wherein catches the furcella clipped form at the GNA agglutinin, then with DEAE and ceramic hydroxyapatite column chromatography. Purifying protein dyes analysis (Figure 56 B) by silver on SDS-PAGE, and also by western blot analysis (Figure 56 C). Make great efforts in early days the spike protein clipped form of energy purifying＞95% purity and reclaim about 50%. The molecular weight of spike protein clipped form is about 170-180kDa.

The total length spike protein uses large quantities of transfection strategies at 293 cells. Expression data prompting is as clipped form, expresses after transfection to reach the highest in about 48 hours and keep stable until 72 hours. Yet, with clipped form and expect on the contrary, full-length proteins is not secreted, but is retained in the cell, as in the Western blotting of cell culture supernatant without any shown in the signal. Albumen total length form is purifying from the cell that Triton X-100 washing agent extracts. Catch the total length spike protein at the GNA agglutinin subsequently, then with hydroxyapatite and SP chromatography. The calculating molecular weight of total length spike protein is about 600kDa, near trimerical Theoretical Mass.

Embodiment 13:SARS virus inoculum

SARS-CoV is only breeding in the Vero cell of check with reference to seed virus, and this virus is used for producing main virus (Master) and work viral seed (Working Virus Seeds) under GMP. Clinical sample from SARS-CoV infected patient respiratory tract is inoculated in the Vero cell of document proof, uses the calibrating culture medium. The culture medium that contains virus is collected and called after 1 (P1) that go down to posterity rear 4 days of infection. The 2nd takes turns virus multiplication carries out in qualified VERO cell again, with the calibrating culture medium, and the P1 virus of 100 times of dilutions of every T-75 flask inoculation 1ml. The culture supernatant rear 3 days of infection collect and be kept at-80 ℃ as P2 with reference to deposit virus, do not carry out plaque purification.

The Vero cell storehouse preparation that is used for further generating SARS-CoV is from the specific cells subgroup, and this subgroup had not been used (as since 1980) since the infectiousness SE occurs. The research cell bank of these cells is with specifying New Zealand's source hyclone preparation. For this research cell bank, master cell bank (MCB) is made under the GMP condition and is only used and specify and control preferably culture medium and additive. Whether the test cell storehouse does not have external reagent, and (" be used for generating the clone feature of biological products " referring to Points To Consider, FDA/CBER 7/1993 according to US applicatory, EU and international guilding principle; ICH Q5D Draft 6 " cell substrate ", on October 23rd, 1996; CPMP/ICH/294/95 " biological technology products quality guideline note: obtain to determine for the cell substrate that generates biotechnology/biological products and feature " (step 4, on July 16th, 97); WHO final draft " zooblast is as the requirement of the external substrate that generates biological products " on March 7th, 1997). This cell bank also needs to test oncogenicity and homogeneity.

Reference virus through plaque purification and in without the qualified Vero cell of FCS amplification with the generation work seed of advocating peace. The selection that another kind helps to guarantee main seed purity and promote safety evaluation is precipitation SARS-CoV and is resuspended in PBS. Viral suspension is made at the most 60% (w/w) sucrose, and candy is arranged, and suspension is transferred to centrifuge tube, covers with 50,40,30 and 20% (w/w) sucrose solution among the PBS. Gradient centrifugation 72 hours, subsequently classification separates. Dilution contains the part of virus and precipitates virion by ultracentrifugation again. Separation is from the RNA of virus precipitation and be transfected in the qualified Vero cell, and wherein " infectivity " positive chain RNA can cause infectious virus to generate, but this virus plaque purification and amplification are to produce the other work of advocating peace seed from purified virus RNA.

Whether the Test Virus seed does not have external reagent (referring to the 21CFR that for example revised on April 1st, 1994, § 630.35, " security test ") and tests homogeneity, uses in certainly independent originate efficient of preparation and antiserum. The viral seed safety property testing that is used for the vaccine purpose is undertaken by service laboratory routine. Wide spectrum PCR test can add test and/or select as another kind of.

Embodiment 14: increase in proportion virus and generate and deactivation

Having enough structural intergrities comprises with generation, deactivation and the purification process that causes the deactivation SARS-CoV that the protectiveness neutralizing antibody reacts in animal model: Vero cell virus infections, and M.O.I. is 0.01, does not have FCS and antibiotic; Collect culture medium, centrifugal removing, whether complete inactivation of property testing is then verified in the BPL deactivation; Filter the deactivation material, carry out MCS-post purifying, be further purified by sucrose gradient centrifugation.

When this basic operation is used for more massive commercial use, can develop some modifications and improvement. At first, can change cell cultivation and infection method and be adapted to roller bottle, be used for preliminary test as intermediate steps in existing BSL 3+ equipment, to produce rapidly. The fermentation process in closed system is used in commodity production usually fully, but roller bottle system can be finished rapidlyer. Roller bottle provides the Vero cell culture systems that truly suspends, and this relative microcarrier is cultivated multiple technologies and security advantages. Suspend to cultivate and to grow into any required fermentation scale, and do not have the closed system of interference cell between going down to posterity, because do not need trypsinized.

For making the infection method in the roller bottle be increased in proportion every batch of 30-50 liter, should at first determine optimum M.O.I. and the collection section of selected culture medium and condition of culture. For larger scale, should use safety to collect and process the method for the high infectious substance of more volume, therefore can be replaced by other method through centrifugal cell separation, as filtering through disposable filter cylinder.

Above-mentioned MCS chromatography and gradient purification step easily are increased to batch volume of 50 liters of as many as in proportion. Yet, for larger scale, for improving purity, use ultrafiltration and aseptic filtration step. Also comprise the nuclease processing of removing host cell DNA.

Embodiment 15: the large scale analysis method

The analytical method of sars coronavirus comprises titration of virus method, immunology and physico-chemical process (Western blotting of the specific corrosioning anteserum of ELISA, PAGE, usefulness antivenom purification intact virus etc.), is used for quantitative purifying antigen and definite feature. Other analytical test comprises: fast output test, by asymmetric territory flow point from detecting and counting with laser particle; Western blotting uses the specific corrosioning anteserum that resists independent virus protein; Be used for the test of residue host cell DNA.

The surplus DNA test is generally finished by hybridization, tests such as operating limit. This test is carried out according to the method for determining and other clone was verified. In addition, can use Threshold^TMMethod.

For generating specific antibody, use all ORFs from SARS-CoV structure and nonstructural gene zone of expression of recombinant proteins. ORFs can clone and be expressed in Escherichia coli, if necessary, and also can be such as baculoviral in eukaryotic vector. This provides the purification of stable albumen of q.s to be used for immune mouse and rabbit with the polyclone and the monoclonal antibody that generate anti-SARS albumen and sets up specific ELISA and measure. Can test different expression vectors with the recombinant protein output of maximization soluble form, different carriers for example, 1 contain the sequence of the terminal histidine residues of 6 N of coding and in addition 1 contain the glutathione-S-transferase albumen that merges SARS PROTEIN C end. Recombinant protein can pass through the single stage column chromatography purification, on nickel chelating agarose or glutathione-sepharose 4B resin. These processes are very fast and generally produce the albumen of 60-90% purity, be applicable to cultivate specific corrosioning anteserum (Pizza etc. (2000) Science 287:1816-20). 5 mouse and 2 rabbits of being used for each recombinant protein can be respectively with 20 and 50 μ g recombinant protein SC immunity, and IFA is as adjuvant, immunity in the 0th, 14 and 28 day. Collected serum with specific the tiring before assessing animal euthanasia at the 7th, 21 and 35 day, animal euthanasia is used for collecting blood and takes out spleen.

For detecting the impurity (for example obtaining the albumen from the Vero cell) in the vaccine product, can use the rabbit anteserum that obtains albumino reaction with Vero-. Obtaining this antiserum is that cell lysate has CFA/IFA by usefulness at least 10 μ g Vero cell lysate immunize rabbits. Can in Western blotting, check serum and Vero-to obtain the reactivity of albumen. For the more specific corrosioning anteserum of the special relevant cell of anti-trend and viral copurification-acquisition albumen, can prepare the simulated infection cell culture that experiences purge process and collect and be used for immunize rabbit.

But development method is determined to tire from immune animal and people's serum neutralization, and does not have to use in the BSL-3+ laboratory restriction of infectious SARS-CoV. A kind of this class strategy is to use recombinant antigen, specificly is spike protein or obtains epitope from furcella that development ELISA measures the antibody that is used for measuring anti-target protein. Suitably epitope can be established correlation in ELISA value and virus and between measured value. The method rapider and more effective (higher yield) is more special tires with protection antibody. This ELISA test also is the ideal tools of monitoring specific antibody in safety testing, must test hundreds of kind animal blood serum in safety testing.

Another kind of strategy be in conjunction with from the structural detail of 2 kinds of pathogenic SARS-CoV and non-pathogenic coronavirus MHV (MHV) to make up the embedded virus like-particles (VLPs) of energy mark. Mensuration be based on the VLPs of octadecyl rhodamine (R18) mark and intercellular fusion (Hoekstra etc. (1984) Biochemistry 23:5675-81). Method depends on that R18 fluorescence self cancellation when merging with cell membrane of including VLPs in alleviates. Coronavirus VLPs display simulation natural viral body relates to their outward appearances in electron microscope (EM) and its BA. Yet, because they do not contain viral RNA, they can not cause subsequently production infection (Vennema etc. (1996) EMBO J 15:2020-2028). The VLP system can be used for MHV (MHV) strains A 59 (MHV-A59) (Godeke etc. (2000) J Virol 74:1566-15), this bacterial strain contains chimeric S albumen. The albumen chimera forms by the extracellular domain of SARS-CoV with from cross-film and born of the same parents' internal area (64 C terminal amino acid residues) of MHV spike protein, chimeric physical efficiency in the OST-7 cell with MHV M (film) and E (coating) albumen coexpression (Godeke etc.). Collect the VLPs that secretes in the supernatant, purifying is also used octadecyl rhodamine (R18) mark (Hoekstra etc.). The VLPs of constant basis is diluted in 96 orifice plates 37 ℃ with continuous serum and hatched 1 hour. Subsequently, add angiotensin invertase 2 (ACE2) in the cell of expression SARS-CoV acceptor, the fusion degree can be used fluorescence spectrophotometer measurement.

Last strategy is that monitoring serum suppresses the interactional ability of iuntercellular cell-Fusion of Cells, cell is the cell and the human cell line who expresses angiotensin invertase 2 (ACE2) of expressing SARS-CoV S albumen, and the latter is the functional receptor (Li etc.) of SARS-CoV. This utilize take reporter gene as the mensuration on basis the fluorescence conversion (green change indigo plant) of fluorogenic substrate CCF2/AM (AM=acetoxy-methyl) when beta-lactamase (Bla) cut as cell-Fusion of Cells read (Zlokarnik etc. (1998) Science 279:84-88). Measure for this, generate the clone that obtains from BHK, its stably express Bla and SARS-CoV albumen. In addition, the human cell line who uses at its surface expression ACE2. Hatched 1 hour with 37 ℃ of the serial dilutions of serum to be tested at its surface expression S albumen and the bhk cell of expressing Bla at kytoplasm. Express the clone of ACE2 and loaded 1 hour at 22 ℃ with 1 μ M CCF2/AM, PBS washes 2 times, cultivates altogether with bhk cell. In the situation of cell-Fusion of Cells, Bla cuts substrate, produces turquoise conversion, excites at 409nm. Therefore, suppress fusion by serum detectable variation is provided.

Embodiment 16: stablize deactivation SARS-CoV

Although the deactivation SARS-CoV vaccine of purifying can be induced the efficient neutralization antibody response in animal, it is relatively unstable and can benefit from preparation to improve the stability of one section allowed time section. Suitably the preparation variation comprises the different buffer solution systems of use, pH scope, stabilising carriers (such as Saccharide and saccharide alcohols, amino acid etc.) etc. Stability test can carry out in real time at normal storage temperature, or carries out with accelerated mode by improving temperature. Therefore, vaccine stability can be increased to about 1 year or longer. The freeze dried vaccine goods also can be used for prolonging self life-span, can increase stability with more additives during freeze-drying.

Embodiment 17: optimization dosage and the arrangement of inactivation of viruses

Report the animal model that SARS-CoV infects, comprised mouse, ferret and macaque. Shown in top embodiment 4, the NAT scope that reaches with the mouse of BPL-SARS-CoV vaccine immunity is 1: 100-1: 1000, and similar with the level of finding among the convalescence patient, and the 100% under fire virus infections that is protected not. Although the mouse challenge model only limits to infect rather than disease, ferret and macaque are useful people SARS disease models. Behind the inoculation SARS-CoV 2 to 4 days, find that ferret and macaque all flow out infectious SARS-CoV particle from throat, nose and pharynx, separating the Vero cell by RT-PCR and/or virus proves. At about same time, it is sleepy that infection animal becomes, and shows respiratory distress and final dead. On histology, SARS-CoV in these animals infects and links from the pulmonary lesion of different severities, with find in biopsy lung tissue and the autopsy material from patient SARS similar. Along with these models can be used, use the research in incubation period of vaccine in mouse, to carry out for immune reading at first, and the effect of optimal dosage and arrangement can be assessed in ferret and macaque model.

Preliminary research in the mouse is used for determining to cause senior middle school and required optimal dosage and the arrangement of antibody horizontal, tires at least in the 1/100-1/1000 scope. Parallel with the active assessment of neutralization, can study the further feature of humoral immune reaction and cell immune response. Can specific assessment from the furcella specific antibody reaction isotype (IgG1 vs.IgG2a) of the serum of immune mouse. Equally, the frequency of spleen CD4+T cellular response BPL-SARS-CoV particle generation IFN-γ and IL-4 can be by ELISPOT and ELISA assessment. These experiments help to see clearly the quality that t cell responses assists to cause the protection antibody reaction.

The vaccine dose that can test increases (from 5 to 20 μ g BPL-SARS-CoV for example, separately or mix equal-volume MF59-citrate) is used SC to anesthetized mice in 100 μ l inoculums. The BALB/c mouse group is strengthened the 0th day sensitization and at the 14th and 28 day, processes 10 at every turn. Relatively the neutralize dynamics that relative furcella specific antibody tires and the Th1/Th2 that assesses Immunel response of secondary terminal point (Secondary endpoint) distributes, therefore after the sensitization the 7th, 21 and 35 day and 2,3,4 and 5 months after the assessment neutralization tire with the furcella specific antibody. After after the sensitization the 21st, 35 day and 2,3,4 and determine after 5 months that IgG2a and the IgG1 of furcella specific antibody tire. The 42nd day and terminal propagation and IFN-γ and the IL-4 generation of assessing the anti-restructuring spike protein of splenic t-cell in 5 months, the restructuring spike protein was from SARS-CoV. The the 7th, 21 and 35 day and 2,3,4 and 5 months after collect peripheral blood. At the 42nd day and 5 months terminal results splenocytes. Determine respectively to neutralize to tire and isotype with the furcella specific antibody by suppressing vero cells infection and ELISA. Proliferation passes through³[H]-thymidine absorbs to be determined. CD4⁺The T lymphocyte generates the frequency of spleen IFN-γ and IL-4 and determines by ELISPOT and facs analysis.

Take the mouse result as the basis, the BPL-SARS-CoV vaccine can be tested in ferret and induce the protectiveness NAT. The immunity ferret is according to similarly arrange and be used in the mouse dosage that caused senior middle school and antibody titer after strengthening for the 2nd time on the 35th day with mouse. 3 groups of ferrets are immune with BPL-SARS-CoV, and independent or mixing equal-volume MF59-citrate is used SC to anesthetized animal in 200 μ l inoculums, process 6 at every turn. Animal is strengthened the 0th day sensitization and at the 14th and 28 day. Collected peripheral blood at the 7th, 21 and 35 day. Determine respectively to neutralize to tire with the furcella specific antibody by suppressing SARS-CoV vero cells infection and ELISA. Each is organized, and ferret avoids infecting for assessment of BPL-SARS-CoV protection immune animal and/or the effect of disease. 2 weeks after strengthening the last time are with 10⁶Intermediate structure is cultivated the (TCID of infective dose unit₅₀) SARS-CoV CDC bacterial strain tracheae in attack anesthetized animal. By from animal, got in rear 20 days in attack nose, pharynx and rectum cotton wipe away to assess SARS-CoV infect (Martina etc., the same). Measure to assess the existence of SARS-CoV in the sample material by RT-PCR and Vero cell infection. The SARS disease clinical symptom of monitor animal, this is by assessing the length of one's sleep, temperature, respiratory symptom, diarrhoea, body weight and survival. Determine protectiveness by viral burst size and duration, disease symptoms duration and severity and surviving animals percentage. Subsequently, can test the preparation that caused senior middle school and antibody titer at the 35th day, the BPL-SARS-CoV that anti-dosage is high 2 times, the latter in same preparation, provide and scheme identical.

Research in addition can be assessed immunogenicity and the effect of candidate vaccine in non-human primate. A 3 compositions year long-tail macaque is immune with BPL-SARS-CoV, and independent or mixing equal-volume MF59-citrate is used SC to anesthetized animal in 500 μ l inoculums, process 4 at every turn. But test b PL-SARS-CoV vaccine is to cause the dosage test of senior middle school and antibody titer on the 35th day after strengthening for the 2nd time in the ferret. Animal is strengthened the 0th day sensitization and in 3 and 6 weeks. Collect peripheral blood in the 1st, 4 and 7 weeks. The Th1/Th2 of secondary terminal point assessment Immunel response distributes. Therefore, tire and peripheral blood CD4+T cellular response restructuring SARS-CoV spike protein generates the frequency of IFN-γ and IL-4 at the 1st, 4 and 7 week assessment neutralizations and furcella specific antibody. Determine respectively to neutralize to tire with the furcella specific antibody by suppressing SARS-CoV vero cells infection and ELISA. Intracellular cytokine dyeing and facs analysis are used for quantitatively generating the CD4 of IFN-γ and IL-4⁺The T cell. Macaque also can protect immune animal to avoid infecting and/or the effect of disease for assessment of BPL-SARS-CoV. 2 weeks after strengthening the last time are with 10⁶Intermediate structure is cultivated the (TCID of infective dose unit₅₀) SARS-CoV CDC bacterial strain attack anesthetized animal, bacterial strain is in the 5ml volume. Several viruses also can be used at conjunctiva, and 0.5ml is in nose and remaining in tracheae. By from animal, got in rear 20 days in attack nose, pharynx and rectum cotton wipe away to assess SARS-CoV infect (Fouchier etc. (200300 Nature 423:240). Measure to assess the existence of SARS-CoV in the sample material by RT-PCR and Vero cell infection. SARS disease clinical symptom that also can monitor animal, this is by assessment length of one's sleep, temperature, respiratory symptom, diarrhoea, body weight and survival. Determine protectiveness by viral burst size and duration, disease symptoms duration and severity and surviving animals percentage.

Mouse

Group	Process	Dosage/approach	The sampling interval	Number of mice
					1-3	BPL-SARS- CoV	20，10，5μg/ SC	7,21,35 days; 2,3,4,5 months;	10 every dosage levels
4-6	BPL-SARS- CoV	20，10，5μg/SC	42 days	10 every dosage levels
					7-9	BPL-SARS- CoV MF59	20，10，5μg/ SC	7,21,35 days; 2,3,4,5 months;	10 every dosage levels
10-12	BPL-SARS- CoV MF59	20，10，5μg/SC	42 days	10 every dosage levels
					13	MF59	NA/ SC	7,21,35 days; 2,3,4,5;	10+10 (killing at the 42nd day and 5 months ends)
14	Salt solution	NA/ SC	7,21,35 days; 2,3,4,5 months;	10+10 (killing at the 42nd day and 5 months ends)

Ferret

Group	Process	By way of	The sampling interval	The ferret number
					1	BPL-SARS- CoV	SC				7,21,35 days	6
2	BPL-SARS-CoV- MF59	SC												7,21,35 days	6
					3	Salt solution	SC				7,21,35 days	6

Macaque

Group	Process	Approach	The sampling interval	The macaque number
					1	BPL-SARS-CoV	SC	Isosorbide-5-Nitrae, 7 weeks	4
2	BPL-SARS-CoV-MF59	SC	Isosorbide-5-Nitrae, 7 weeks	4
					3	Salt solution	SC	Isosorbide-5-Nitrae, 7 weeks	4

Embodiment 18: human T-cell's reaction

As the pioneer that people's clinical research begins, have by oneself different HLA haplotypes healthy donors periphery blood T lymphocyte reactive available external sensitization technology evaluation (Abrignani etc. (1990) Proc Natl Acad Sci US A87:6136-40). The purpose of this research is the immunodominance T cell antigen epitope of indicating at first in the SARS-CoV albumen. Briefly, there is the PBMCs of the healthy donors of different HLA haplotypes in the culture medium that contains 5% autoserum, to cultivate from 20, has the SARS-BPL-CoV particle of variable concentrations, scope from 0.5 to 20 μ g/ml. The expression of assessment activation mark after 24 and 48 hours. Cultivate and assessed afterwards the T lymphocyte frequency that generates IFN-γ and IL-4 in 12 hours and 15 days, have the 100U/ml recombinant human il-2. Classification is selected and is generated CD4T lymphocytes activation cell factor and finally as single cell clone, use the FACS technology. Having the human experimenter's of different HLA CD4+T cell repertoire by oneself assesses by the clone's of CD4+T clone and anti-self EBV-transformation cell lines proliferation assay, transformation cell lines loads the stack peptide of 15 aggressiveness, and peptide is from dependency structure and the non-structural protein of SARS-CoV.

When turning to the actual persons test, security and the immune response in the assessment normal adults after the immunity in muscle, the BPL-deactivation SARS-CoV vaccine that increases dosage is used in immunity, comprises MF59 adjuvant or omission, and this depends on clinical data. In the 1st group, gave 3/4 immunity at 0,1,6 month, in the 2nd and the 3rd group, respectively 0,1,2,6 month and 0,2,6 weeks gave immunity. Test is adopted blindly and is observed, and with placebo in contrast. The experimenter is divided into each dosage level at random. Immune response parameter to be measured comprises serum neutralizing antibody, ELISA antibody and generates the peripheral blood CD4+T cell of IFN-γ, dyes by intracellular cytokine.

Group	Antigen dose (μ g)	Use arrangement	Experimenter's quantity for the treatment of	Experimenter's quantity with placebo	The sampling interval
						A1
	10	0,1,6 months	18	6	0,1,2,6,7 months
						A2
	20	0,1,6 months	18	6	0,1,2,6,7 months
						B1
	10	0,1,2,6 months	18	12	0,1,2,6,7 months
						B2
	20	0,1,2,6 months	18	12	0,1,2,6,7 months
						C1
	10	0,2,6 weeks	18	12	0,2,6,10,30 weeks
						C2
	20	0,2,6 weeks	18	12	0,2,6,10,30 weeks

Embodiment 19: select to be used for the Chinese hamster ovary celI system that spike protein is expressed

The method of better having established acquisition Chinese hamster ovary (CHO) clone is used for HIV and HCV, this clone stably express viral envelope glycoprotein, complete, the suitable glycosylation of protein conformation and effectively in conjunction with neutralizing antibody (Srivastava etc. (2002) J Virol 76:2835-47; Srivastava etc. (2003) J Virol 77:11244-259; Heile etc. (2000) J Virol 74:6885-92). Identical technology can be applied to SARS-CoV, for generation of 2 kinds of different stable CHOK-1 clones, generates total length or brachymemma SARS spike protein. Spike protein can be used construction expression described herein, but does not have the 6-His mark. Can compare these albumen generate neutralizing antibody in immune animal ability and their expressions in the CHOK-1 cell.

A pCMV3 carrier of expressing furcella can be used for obtaining to stablize CHOK-1 clone, and clone contains the DHFR of cmv enhancer/promoter, ampicillin resistance, fusion and is used for selecting purpose reduction neomycin gene. Stable cell lines can generate in the CHOK-1 cell with neomycin base selective system. The integrality of clone to confirm to insert can check order, can use Trans-LT1 polyamine transfection reagent (PanVera Corp., Madison, WI) carry out transient transfection with the assessment expression and also can express protein integrity by ELISA and western blot analysis assessment.

Initial Chinese hamster ovary celI is chosen to pollute risk informed associated adjustment standard without TSE/BSE. For making up clone, process comprises transfection, uses the selective medium Preliminary screening, and then subclone is to guarantee clone purity. Available antigen capture ELISA measures cell conditioned medium with quantitative all selections and the expression in amplification stage. Express for the total length furcella, by the internal representations of immunofluorescence dyeing screening methyl alcohol fixed cell, use the anti-SARS antibody of rabbit. Can be used for guaranteeing expression in T-75 flask amplification stage continuous measurement. The molecular weight and the integrality that check expressing protein can by the PAGE under natural and reduction and the Denaturing, then be that immunity is surveyed.

The pCMV3 carrier of expressing total length or clipped form SARS-CoV spike protein can import the CHOK-1 cell, uses Trans-LT-1 reagent and Nonsele ctive culture media. After the transfection 24-48 hour, depend on cell density, cell becomes the selective medium that contains 500 μ g/ml neomycins with ratio division in 1: 5 and culture medium. Any cow's serum that uses in these processes meets adjustment criteria from without the TSE source. After 10 to 14 days, choose independent bacterium colony and transfer to 96 orifice plates, cultivating in the Nonsele ctive culture media fully. When about 80% hole was paved with, 24 hours supernatants can be caught ELISA by furcella and be screened. Be initial expression total length spike protein, cell can be fixed and by immunofluorescence dyeing screening, uses the anti-SARS antibody of rabbit with methyl alcohol. After the low expression of the removal clone, clone is less than 20-30, catches the expression after ELISA and Western blotting can be used for definite lysis subsequently. Each clone of a precipitable part is weighed and cracking in the 1%Triton lysis buffer, is used for determining expression. 3 to 4 clones that generate the correct spike protein of highest level structure and conformation can expand 3 liters of bioreactors to and adapt to and be used for the low serum condition of suspension culture that enlarges.

The antigen capture ELISA that is used for the SARS spike protein measures available 96 hole flat undersides and carries out, the purifying immunoglobulin (Ig) in the coated every hole of 250ng of plate, and immunoglobulin (Ig) obtains the rabbit anteserum of personal deactivation of SARS virus immunity. Add supernatant or lysate sample and hatched 2 hours at 37 ℃. The SARS that conjugated antigen compiles relatively^+veSerum or high-affinity monoclonal antibody reactive, monoclonal antibody are the anti-SARS spike proteins of people or mouse, with the two anti-detections of suitably planting special peroxidase conjugated. Plate tmb substrate (Pierce, Rockford, IL) colour developing, in the 450nm wavelength readings, the protein concentration of every ml sample obtains from calibration curve (OD vs. protein concentration), and calibration curve is take the serial dilution of the restructuring spike protein of concentration known as the basis.

Also carry out immune detection analysis, the standard method of following is described in Srivastava etc. and (2002) are the same. Briefly, 10-20 μ l sample is analyzed at 4-20%SDS PAGE, under the Denaturing of non-reduced/appropriate heating. Subsequently, protein delivery reacts to nitrocellulose membrane and with Anti-TNF-α furcella rabbit anteserum, then is the anti-rabbit Ig that puts together Alexa 688 (Molecular Probes, Oregon). With infrared imagery system scan point.

The spike protein that the candidate cell that screens high expressed ties up in (3 liters) perfusion bioreactor is on a small scale expressed and stability. Further expression and the expressing protein integrality of assessment candidate clone are tested expression stability subsequently in nonoptional situation. Test selected clone and keep the situation of integrating SARS spike protein gene DNA sequence integrality. Be Fast Monitoring bottle and 3 liters of expressions of assessing in cultivating, the method (Gluvanthus Nivalis agglutinin) that has developed based on agglutinin arrives the certain purity level for separating of the SARS spike protein, and this level allows the albumen in sxemiquantitative CHO supernatant and determines feature. The total length spike protein obtains from the cell that Triton X-100 washing agent extracts, and catches at the GNA agglutinin subsequently, then with hydroxyapatite and SP chromatography. Then determine the feature of eluted protein, by: (1) polyacrylamide gel electrophoresis (PAGE) and coomassie dyeing, (2) survey with the immunity of anti-SARS rabbit anteserum, (3) are determined with the architectural feature of size exclusion chromatographies (SEC) and with the mass spectral analysis of MALDI-TOF.

The productivity of expressing the Chinese hamster ovary celI system of SARS spike protein should be at least 2mg/L and be the 3mg/100gm cell for the total length spike protein, and cell is in steady state cell density. From 45 days, the output of 2.5 liters of bioreactors be ~ the 1000mg crude protein.

Embodiment 20: purifying is used for the spike protein of people's vaccine

For the purpose of purifying SARS spike protein for generation of human GMP level material, use following basic skills, carry out at 2-8 ℃ in steps: initial substance is the Chinese hamster ovary celI culture supernatant (20-30X) that concentrates, and thaws and through 0.45 μ m membrane filtration; This material is severe contamination albumen and the DNA from culture; The 1st purification step is the affinity chromatography of using Gluvanthus Nivalis (GNA), and GNA is the agglutinin of preferably identifying the terminal mannose of carbohydrate containing; Catch glycosylated protein, comprise the SARS spike protein, non-glycosylated protein and DNA be not in conjunction with this post; The GNA post then carries out 2 with the chromatographic step of circulation pattern operation; Anionite DEAE and ceramic hydroxyapatite (cHAP); DEAE pollutes upper albumin and DNA in conjunction with some, and cHAP is in conjunction with polluting arbitrarily haemocyanin; Total length spike protein purifying from cell precipitation; Cell is caught the total length spike protein at the GNA agglutinin subsequently with Triton X-100 cracking, then is hydroxyapatite and SP chromatography.

Can further process the SARS furcella of purifying to remove adventitious viruses: at 3.51 hours inactivation of viruses of pH; Subsequently concentrating sample and diafiltration caught purifying protein with the SP resin at last in the buffer solution of pH 4; Spike protein flows through in conjunction with this resin and a lot of virus.

The wash-out spike protein, concentrated also diafiltration is in buffer solution. Large quantities of products of making are followed through 0.2 μ m membrane filtration with by DV50 virus removal membrane filtration. Large quantities of materials of making are filled in the appropriate containers, for example 3.0ml bottle, 100 grade laminar flow hoods.

The method of testing of each purification step comprises that albumen is concentrated, endotoxin (LAL), biological load and recovery.

Before the people uses, render a service test and can assess vaccine affects specific reaction in external or in vivo studies specifiability. Determine that immunogenicity in the body is quantitative group by 10 mouse, with the proteantigen of various dose. Exist with the IgG antibody in the elisa assay serum. The standard of passing through is based on seropositive vaccine and processes size of animal, compares with normative reference. Other test comprises overall security, aseptic, purity, vaccine homogeneity (use is specific to the ELISA of spike protein), amount albumen concentrated (UV spectrophotometric absorption process is take the molar absorptivity rate of aromatic amino acid as the basis).

Large quantities of drug substances and whole container product are carried out stability test. Assess large quantities of products and be the temperature at-60 ℃ (recommendation conditions of storage), 25 ± 2 ℃ and 40 ± 2 ℃, protection is not subjected to illumination, and time point is 0,3,6,9 and 12 months. Whole container product is in temperature-60 ℃ test, and reverse 5 ± 3 ℃, 25 ± 2 ℃ and 40 ± 2 ℃, time point is 0,3,6,9,12 month. The stability indication is measured and can be comprised outward appearance, pH, protein content, SDS-PAGE, size exclusion HPLC, container/closure integrity, and the simple sample of testing in large quantities of and triple whole container contents carries out.

The albumen of the method purifying can be assessed in mouse, rabbit and ferret, as mentioned above, and take top embodiment 4,5,8 and 9 result as the basis.

Initial experiment is carried out determining to cause senior middle school and required GMP spike protein optimal dosage and the arrangement of antibody horizontal in mouse, is tired at least in the 1/100-1/1000 scope. Spike protein is tested in the scope of 5 to 40 μ g, independent or mixing equal-volume MF59-citrate, anesthetized mice in 100 μ l inoculums. Immunity BALB/c mouse group is processed 10 at every turn. Animal is strengthened the 0th day sensitization and at the 14th and 28 day. The secondary terminal point dynamics that relative furcella specific antibody is tired that relatively neutralizes distributes for assessment of the Th1/Th2 of Immunel response. After the sensitization the 7th, 21 and 35 day and 2,3,4 and 5 months after assessment neutralization tire with the furcella specific antibody; After after the sensitization the 21st and 35 day and 2,3,4 and determine after 5 months that IgG2a and the IgG1 of furcella specific antibody tire; The 42nd day and terminal propagation and IFN-γ and the IL-4 generation of assessing the anti-restructuring spike protein of splenic t-cell in 5 months, the restructuring spike protein was from SARS-CoV; The the 7th, 21 and 35 day and 2,3,4 and 5 months after collect peripheral blood; At the 42nd day and 5 months terminal results splenocytes; Determine respectively to neutralize to tire and isotype with the furcella specific antibody by suppressing SARS-CoV vero cells infection and ELISA. Proliferation passes through³[H]-thymidine absorbs to be determined. CD4⁺The T lymphocyte generates the frequency of spleen IFN-γ and IL-4 and determines by ELISPOT and facs analysis.

Secondly, determine optimal dosage and the arrangement of restructuring spike protein in ferret. Take the mouse result as the basis, cause the high 2 times restructuring spike protein test of furcella vaccine relative dosage that the highest antibody neutralization is tired, the restructuring spike protein provides in same preparation. The SC immunity under with 200 μ l inoculum narcosises of 3 groups of ferrets is processed 6 at every turn. Animal is strengthened the 0th day sensitization and at the 14th and 28 day. Collected peripheral blood at the 7th, 21 and 35 day. Determine respectively to neutralize to tire with the furcella specific antibody by suppressing SARS-CoV vero cells infection and ELISA. Similar with the research of the ferret of front, each is organized, and ferret avoids infecting for assessment of vaccine protection immune animal and/or the effect of disease.

The immunogenicity of candidate vaccine and effect also can be assessed in non-human primate. The long-tail macaque restructuring SARS-CoV spike protein immunity of 3 composition years, independent or mixing equal-volume MF59-citrate is used SC to anesthetized animal in 500 μ l inoculums, process 4 at every turn. Test spike protein vaccine is to cause the dosage test of senior middle school and antibody titer on the 35th day in the ferret. Animal is strengthened the 0th day sensitization and in 3 and 6 weeks. Collect peripheral blood in the 1st, 4 and 7 weeks. The Th1/Th2 of secondary terminal point assessment Immunel response distributes, as mentioned above (neutralization is tired with the furcella specific antibody, and peripheral blood CD4+T cellular response restructuring spike protein generates the frequency of IFN-γ and IL-4, in the assessment of the 1st, 4 and 7 weeks).

At last, there is the IM restructuring SARS vaccine of MF59 adjuvant to carry out people's stage, placebo, dosage increase, security/Study On Immunogenicity. Security after the test assessment immunity in the normal adults and immune response, the SARS recombinant vaccine that increases dosage is used in immunity, and vaccine has the MF59 adjuvant, uses in the muscle. Gave 3/4 immunity at 0,1,6 month. Test is the blind and placebo of observer. The experimenter is divided into each dosage level at random. Immune response parameter to be measured comprises serum neutralizing antibody, ELISA antibody and generates the peripheral blood CD4+T cell of IFN-γ, dyes by intracellular cytokine:

Group	Vaccine antigen dosage (μ g)	Use arrangement	Experimenter's quantity for the treatment of	Experimenter's quantity with placebo (MF59)	The sampling interval
						A1
	50	0,1,6 months	18	6	0,1,2,6,7 months
						A2
	100	0,1,6 months	18	6	0,1,2,6,7 months

Embodiment 21: compare inactivation of viruses and purifying spike protein

Immunogenicity and the effect that can in non-human primate, compare inactivation of viruses and purifying spike protein. 3 compositions year long-tail macaque is used from the restructuring SARS-CoV spike protein of Chinese hamster ovary celI system or immune with BPL-SARS-COV, 4 of each processing, used dosage and preparation in front immunogenicity are attacked and to be caused senior middle school and antibody titer in the experiment, use SC to anesthetized animal in 500 μ l inoculums. Animal is strengthened the 0th day sensitization and in 3 and 6 weeks. Collect peripheral blood in the 1st, 4 and 7 weeks. The Th1/Th2 of secondary terminal point assessment Immunel response distributes, as mentioned above.

Group	Process	Dosage/approach	The sampling interval	The macaque number
					1	The Rec-spike protein+or-MF59	Yμg/SC	Isosorbide-5-Nitrae, 7 weeks	4
2	BPL-SARS-CoV+or-MF59	Yμg/SC	Isosorbide-5-Nitrae, 7 weeks	4
					3	Salt solution	NA/SC	Isosorbide-5-Nitrae, 7 weeks	4

Embodiment 22: express in yeast

Yeast is useful and cheap eukaryotic expression system. The albumen of Yeast expression is used for the recombinant hepatitis B virus vaccine, and restructuring SARS antigen also can always be expressed at yeast, is used for vaccine use. Yeast expression generates also more convenient for antigen, antigen is for the preparation of monoclonal and polyclonal antibody, or is used for determination of serology.

Clone's nucleocapsid protein (N) and from 2 multi-form spike glycoproteins (S) of sars coronavirus FRA bacterial strain (AY310120) is used for expressing at saccharomyces cerevisiae (S.cerevisiae):

SARS N: amino acid/11-422 (the coordinate 28120-29388 of AY310120 bacterial strain)-Figure 65

SARS furcella: amino acid/11 4-1195 (membrane spaning domain and kytoplasm tail disappearance)-Figure 66

SARS furcella: amino acid/11 4-662 (S1 domain)

Be to generate S1 and make up, the XhoI-NotI fragment of about 3733bp is as starting point, its total length spike glycoprotein of encoding. PCR is used for minute XbaI-BlnI of 2 sections amplification full-length gene: 2440bp and the BlnI-SalI of 1306bp. These fragments are subcloned into respectively (Novagen): pT7Blue2XbaI-BlnI in the commercial carrier (5 ' end of spike glycoprotein) and pT7Blue2BlnI-SalI (3 ' end of spike glycoprotein; Figure 58). Following primer is used for PCR reaction subsequently: Spk-1 (5 ') SEQ ID NO:9785; Spk-2 (5 ') SEQ ID NO:9786; Spk-3 (5 ') SEQ ID NO:9787; Spk-4 (5 ') SEQ ID NO:9788.

The Escherichia coli HB101 competent cell transforms PCR and connects product and containing the Luria agar plate upper flat plate cultivation of 100 μ g/ml ampicillins. Required clone selects DNA analysis to identify with Microtraps. Behind sequence affirmation and the required subclone of plasmid amplification, need to remove the SalI site, inside that exists in the furcella sequence X baI-BlnI part, be cloned in the Yeast expression carrier (BamHI-SalI) to help future. Therefore, we prepare the CelII-MfeI carrier contains the SalI site with removal 143bp sequence from pT7Blue2XbaI-BlnI (5 ' terminal furcella). The oligonucleotides DS1-6 of kinases (SEQ ID NOS:9789-9794) is connected into the CelII-MfeI carrier subsequently to replace the 143bp (changing without amino acid) that is used for sudden change SalI site and removes, and produces pT7Blue2.XbaI-BlnI Δ sal.

5 ' XbaI-BlnI (from pT7Blue2.XbaI-BlnI Δ Sal) and 3 ' BlnI-SalI (from pT7Blue2 BlnI-SalI) spike glycoprotein are inserted through gel-purified, they are connected into p893-1XbaI-SalI carrier (obtain the carrier from pLitmus 38 (New England Biolabs), α factor targeting sequencing is cloned into the BamHI-Sal I site of MCS). Gained total length SARS furcella coded sequence called after p893-1.SARS Spike 1255 #9 (Figure 58).

The Escherichia coli HB101 competent cell transforms oligonucleotides and replaces the connection product and containing the Luria agar plate upper flat plate cultivation of 100 μ g/ml ampicillins. Required clone selects DNA analysis to identify with Microtraps. After confirming the positive colony sequence, select pT7Blue2 Xba-Bln Δ Sal as the PCR reaction template with amplification furcella S1 1967bp Xba-Sal fragment. Fragment is subcloned in the p893-1Xba-Sal carrier subsequently, confirms sequence, and called after p893-1.Spike S1#11 (Figure 59).

For being cloned among the saccharomyces cerevisiae expression pBS24.1, S1 sequence 5 ' end must be modified into HindIII from XbaI, is connected with ADH2/GAPDH BamHI-HindIII promoter fragment with permission ' the terminal connection of HindIII. Gel-purified AgeI-SalI 1943bp fragment from pT7Blue2Xba-Bln Δ Sal (above-mentioned). The oligonucleotides of this fragment and HindIII-AgeI 30bp zymogenesis (S1-1+S1-2 produces 5 ' essential HindIII site) is synthetic to being connected into together commercial subcloning vector (the called after pSP72.SARS Spike S1 #2 of pSP72HindIII-SalI; Figure 59). S1-1 has SEQ ID NO:9795 and S1-2 has SEQ ID NO:9796.

The sequence of positive colony of DNA analysis is selected in affirmation from Microtraps after, gel-purified HindIII-SalI fragment. 1365bp BamHI-HindIII ADH2/GAPDH promoter fragment is connected into pBS24.1 BamHI-SalI carrier with 1973bp HindIII-SalI S1 fragment, produces the upper pd.SARS furcella S1#2 expression plasmid (Figure 60) of transforming of heredity.

Wine brewing yeast strain AD3 transforms pd.SARS furcella S1#2, checks that single transformant glucose in culture medium exhausts expression afterwards. Detected such as Coomassie blue stain, recombinant protein is high level expression in yeast. The specific conversion of yeast cells SARS S1 expression plasmid uses Invitrogen S.c.EasyComp^TMThe conversion reagent box. Expression is shown in Figure 57.

Furcella 1195 albumen do not contain cross-film (TM) district or the kytoplasm tail that exists in the total length SARS structure, for expressing this albumen, carry out following a series of genetic manipulation:

Gel-purified BlnI-DraI 1056bp fragment from pT7Blue2BlnI-SalI#11 (above-mentioned). Oligonucleotides (the DRS1+2 of this fragment and 68bp DraI-SalI zymogenesis; SEQ ID NOS:9797 ﹠ 9798) synthetic to being connected into together pT7Blue2BlnI-SalI carrier (Figure 61). The Escherichia coli HB101 competent cell transforms oligonucleotides and replaces the connection product and containing the Luria agar plate upper flat plate cultivation of 100 μ g/ml ampicillins. Required clone selects DNA analysis to identify with Microtraps. After sequence is confirmed, clone's called after pT7Blue2 BlnI-Sal furcella 1195 #7. The 1126bp BlnI-SalI fragment of gel-purified coding furcella 11953 ' end.

Be to produce XbaI-SalI furcella 1195 fragments, 3109bp XbaI-PciI fragment is separated from p893-1.SARS furcella 1255#9 (above-mentioned) and 457bp PciI-SalI fragment fragment from pT7Blue2.SARS furcella 1195 #7 (above-mentioned). 2 fragments are cloned in the p893-1XbaI-SalI carrier, produce p893-1.SARS furcella 1195 #34 plasmids (Figure 62).

In clone SARS furcella 1195 to pBS24.1 saccharomyces cerevisiae expressions, SARS furcella 11955 ' end must be modified into HindIII from XbaI, do such as above-mentioned furcella S1 expression cloning. At first, 2416bp AgeI-BlnI fragment is separated from p893-1.SARS furcella 1195#34. This fragment is connected into pT7Blue2 HindIII-BlnI carrier together with the HindIII-AgeI 30bp oligonucleotides that synthesizes (produce as mentioned above S1 albumen, be used for expressing at saccharomyces cerevisiae). The Escherichia coli HB101 competent cell transforms oligonucleotides and replaces the connection product and containing the Luria agar plate upper flat plate cultivation of 100 μ g/ml ampicillins. Required clone selects DNA analysis to identify with Microtraps. After confirming positive colony sequence and plasmid amplification pT7Blue2.SARS 11955 ' HindIII-BlnI#10 (Figure 63), we isolate 402bp HindIII-NcoI fragment and 2044bp NcoI-BlnI fragment (Figure 63). HindIII-BlnI separate to need to finish in 2 steps, and avoiding relating to clone's event in inner HindIII site, this site is positioned at the nucleotides numbers 1319 of furcella 1195 albumen.

For the BamHI-SalI expression cassette with furcella 1195 is assembled in the pBS24.1 carrier, the Escherichia coli HB101 competent cell transforms BamHI-HindIII (ADH2/GAPDH promoter), HindIII-NcoI 402bp fragment, NcoI-BlnI 2044bp and BlnI-SalI 1126bp fragment and is transformed in the pBS24.1BamHI-SalI carrier. Sample is cultivated at the Luria agar plate upper flat plate that contains 100 μ g/ml ampicillins. Required clone identifies with little screening DNA analysis, thereby produces the pd.SARS furcella 1195#10 (Figure 64) of genetic modification.

Wine brewing yeast strain AD3 transforms pd.SARS furcella 1195#10, checks that single transformant glucose in culture medium exhausts expression afterwards. Recombinant protein detects by Coomassie blue stain. The specific conversion of yeast cells SARS S1 expression plasmid uses Invitrogen S.c.EasyCompTM conversion reagent box.

Embodiment 23: express in mammal cell line

The cDNA fragment that contains S albumen ORF increases from the SARS virus RNA (Frankfort separator) that grows in the Vero cell by RT-PCR, and S albumen ORF has 1255 amino acid. The PCR fragment of amplification is cloned in the pBlueScript carrier, and order-checking is assembled total furcella sequence to produce total length SARS furcella clone pBSnSh. In-vitro transcription pBSnSh then translates in the rabbit reticulocyte lysate, produces the single polypeptide of estimating molecular weight ~ 140kDa.

This plasmid insert through XhoI and Not I be cloned into again mammalian expression vector pCMVIII (Srivastava etc. (2003) J.Virol.77:11244-11259) make up nSh (Figure 74 A) to produce. The PCR fragment comprises 1195 amino acid whose spike proteins of tool, its disappearance cross-film (TM) domain and the abundant kytoplasm tail (Cy) of cysteine, and this fragment that increases is also cloned the pCMVIII carrier to produce structure nSh Δ TC (Figure 74 B). 2 kinds of structures all use 6 histidine residues at the C end mark, are used for assist feature and determine. XhoI/NotI fragment without histidine mark is subcloned into Alphavirus replicon carrier main chain pVCRchim2.1, is used for generating the Alphavirus replicon particle chimera of expressing S albumen. The generation of replication defect type Alphavirus carrier particle and feature are definite carries out substantially as previously mentioned (Perri etc. (2003) J.Virol.77:10394-10403; Polo etc. (1999) PNAS USA.96:4598-4603). Gained Alphavirus carrier particle called after VEE/SIN.

COS7 cell and BHK-21 cell maintain in the Dulbecco improvement Eagle culture medium that adds 10% hyclone, 5%CO in 37 ℃ and the air₂ COS7 cell transfecting expression plasmid (nSh, nSh Δ TC) uses transfection reagent box (TransIt-COS, Mirus), follows manufacturer's explanation. Cell washes 1 time and uses 1x lysis buffer (20mM MOPS, 10mM NaCl, 1.5mM MgCl with ice PBS₂With 1%Triton X-100) cracking, buffer solution contains intact proteins enzyme inhibitor (Roche). After hatching 30 minutes on ice, centrifugal removing fragment. Clarify lysate or purifying or be directly used in Western blotting.

Be purifying secreted spike protein, from transfectional cell, collect culture medium and with centrifugal 10 minutes of 12,000rpm to remove cell fragment. The clarification culture medium is applied to ConA-agarose column (Vector Lab). Post is fully washed with the 20mM sodium phosphate buffer, uses subsequently 1M methyl α in the 20mM sodium phosphate buffer-D-mannopyranose glycosides (MMP), 1M NaCl elution of bound albumen. Contain the post certain applications of SARS-CoV spike protein in MagneHis protein purification system (Promega), follow operation recommended by the manufacturer.

For western blot analysis, by the 4-20%SDS-PAGE protein isolate and thereupon electrophoretic transfer to nitrocellulose membrane (Invitrogen). Film is sealing in sealing buffer solution (5% skimmed milk and 0.1% soil temperature 20 among the PBS), with indicating the antibody incubated at room 1 hour, wash and survey with two anti-(Biosource) that horseradish peroxidase (HRP) is puted together, (the ECL system is Amersham) with the exposure X-ray film in then chemiluminescence. Used antibody is the anti-histidine antibody of mouse monoclonal (anti-His mark monoclonal antibody, Novagen), the anti-peptide antibody of the rabbit polyclonal of anti-SARS-CoV spike protein (SmPab, Abgent) or the anti-SARS serum of rabbit (2BE) by obtaining with purifying SARS-CoV virion immunize rabbit. It is 1/2,500 that the latter's cell culture neutralization is tired. Except as otherwise noted, for anti-histidine antibody and SmPab, antibody uses with 1/1,000, and for anti-SARS rabbit anteserum, antibody uses with 1/10,000.

Some spike proteins are processed with peptide-N glycosidase F (PNGase F). Cell lysate dilutes in 0.5%SDS and 1% β-mercaptoethanol and 100 ℃ of sex change 10 minutes. After 2 times of 1%NP-40 dilutions in the 50mM sodium phosphate (pH 7.5), sample was processed 1 hour with 37 ℃ of PNGase F (NEB). Enzyme is processed sample and is analyzed under reducing condition by 4-12%SDS-PAGE. For the part digestion with PNGase, cell lysate was processed 3 hours with 50mM sodium phosphate (pH 6.0) dilution that contains 0.75%Triton-X and with 37 ℃ of PNGase F (Calbiochem). Enzyme is processed sample and is analyzed under non-reduced condition by 4-20%SDS-PAGE.

48 hours cell protein trace is shown in Figure 75 after the transfection. When analyzing when the boiling lysis thing and under reproducibility SDS-PAGE condition, detect S albumen doublet in the cell lysate, estimate molecular weight ~ 170 ~ 180kDa (Figure 75 A, swimming lane 3). Produce this doublet seemingly since behind PNGase F pretreatment cell lysate the different glycosylation of 1 peptide species product, PNGase F is reduced into ~ single kind (Figure 75 A, swimming lane 4) of 140kDa doublet. This is the expection size from total length, the prediction of complete polypeptide product amino acid sequence. This experiment show total length SARS-CoV in mammalian cell, be expressed as single, do not cut polypeptide, but 2 kinds of different glycosylation forms are arranged, be respectively gp170 and gp180. 2 S sugar types unlike full length sequence coding are not secreted, and all detect in cell lysate (Figure 75 A, swimming lane 5) and cell culture medium (Figure 75 A, swimming lane 3) ~ the S Δ protein product of 160kDa single kind.

Be the feature of further determining to process in the S albumen born of the same parents, as mentioned above, infect the BHK21 cell with the deficiency Alphavirus particle of expressing total length S. After infecting 6 hours with MOI 5, infection cell L-[³⁵S] methionine/cysteine pulse labeling 1 hour and following the trail of 2 or 4 hours. Immunoprecipitation [³⁵S] the S albumen of mark, the rabbit anti-serum that uses anti-deactivation, purified virus to cultivate, subsequently Endo H digestion. Endo H processing comprises with sample buffer (50mM sodium phosphate, 0.1%SDS, 50mM DTT, pH 6.0) diluted and boils 5 minutes. After the sex change, further with 0.75% Triton-X 100 dilution with follow manufacturer's explanation (Calbiochem) and processed 3 hours with 37 ℃ of endoglycosidase H (Endo H). Enzyme is processed sample and is added the sample loading buffer that contains 0.1%SDS and DTT, and 8%SDS-PAGE analyzes.

Digestion and not digestible protein all in SDS, boil and analyze (Figure 55) by reproducibility SDS-PAGE. After the pulse in 1 hour, S albumen occurs with the gp170 single component, to Endo H responsive (swimming lane 1 and 2). After the pulse in 2 hours, new kind (gp180) (swimming lane 3) occur together with about equal proportion and gp170. After the pulse in 4 hours, the gp180 kind is main S protein ingredient (swimming lane 5), present anti-Endo H (swimming lane 5 and 6). These data and gp170 contain the resident glycoprotein of ER-of high mannose chain and gp180 to there being the golgiosome processed sugar albumen that contains Endo H resistance composite oligosaccharide consistent.

Also test the Endo H sensitiveness of the C terminal deletion S Δ albumen of purifying from cell culture medium. Shown in Figure 76, the S Δ of finding to observe in the cell lysate is to Endo H responsive (swimming lane 1 and 2), and the anti-Endo H of secretion S Δ (swimming lane 3 and 4) in the cell culture medium. This result and this glycoprotein are synthetic consistent in ER with the prematurity form before transferring to golgiosome, and golgiosome adds complex carbohydrate and secretory protein subsequently.

As mentioned above, detect the S albumen of COS7 cells in the cell lysate western blot analysis, it is with gp170/gp180 doublet form, and cell lysate is by boiling abundant sex change when DTT exists. Yet when the same cell lysate did not have thermal denaturation before with the SDS-PAGE western blot analysis, the most of S albumen that detects was the macromolecule glycoprotein (Figure 77, swimming lane 1) in the 440-669kDa scope. The anti-10mM DTT of ~ 500kDa kind processes (swimming lane 3) and is not dissociated into single aggressiveness form, unless lysate is first at 100 ℃ of thermal denaturations (swimming lane 4). On the contrary, test proteins (thyroglobulin) the oligomerization form kept by disulfide bond of quaternary structure is processed by 10mM DTT and is transformed into the subunit form. These Notes of Key Datas S albumen ~ 500kDa oligomerization form is not that disulfide bond connects and thermally labile. For determining the heat sensitivity of S albumen ~ 500kDa kind, repeat the thermal denaturation experiment, but do not have DTT. Shown in Figure 78, be enough to make it to be transformed into the single aggressiveness form (swimming lane 4) of gp170/180 at 100 ℃ of independent thermal denaturation ~ 500kDa albumen. Use 80 ℃ of thermal denaturation steps, detect be in similar proportion ~ 500kDa and single aggressiveness form.

For further research this ~ whether the 500kDa kind represent the S albumen oligomer of native conformation, with obtaining from the comparative analysis of the S of virion glycoprotein, this albumen is from the Vero cell culture. Behind the SDS PAGE, the purified virus body is dissolved in 1%SDS, then carries out western blot analysis. In the virion particle, confirm existence ~ 500kDa spike protein oligomer (Figure 79, swimming lane 1). In addition, the oligomer that produces of thermal denaturation dissolubility virion changes and with total length identical (swimming lane 2,3) that S observes of recombinating to single aggressiveness. In crosslinked experiment, further analyze the oligomerization character of virion S. (pH 8, and Sigma) dilution is 2 times with the processing of 1% final concentration and with 0.2M triethanolamine-HCl from saccharose gradient five equilibrium inactivation of viruses body and function 10%SDS partly; Add subsequently the dimethyleneimine (DMS from fresh preparation solution (10mg/ml is in 0.2M triethanolamine-HCl); Pierce Chemical Co.), final concentration is 3.3mg/ml. After the room temperature 2 hours, sample is concentrated with Centricon-30, dyes analysis by silver behind 4% polyacrylamide gel electrophoresis. Be untreated and the crosslinked virion albumen of DMS thermal denaturation all, dye the effect (Figure 80) of heat to keeping the oligomerization structure of analyzing by SDS-PAGE and silver. Do not have when crosslinked, thermal denaturation causes single aggressiveness kind replacement ~ 500kD spike protein kind. On the contrary, in crosslinking protein, ~ 500kD and single aggressiveness kind significantly do not change after heating. The oligomer of the single glycoprotein polyprotein precursor of the covalently bound S of these Data supports ~ 500 kD albumen right and wrong. Crosslinked and boil after, ~ 500kD kind moves, and is slower than the form diffusion of being untreated. It may be owing to boil the structural change that causes that this mobility changes. In addition, can find ~ the small protein kind of 300kD that it may represent the non-S of dissociating dimer.

Be the size of the restructuring of more accurate estimation COS7 cells ~ 500kDS kind, the COS7 cell lysate that contains S albumen oligomer separates with the classification of size exclusion column chromatography. The major part of ~ 500kD oligomer 572kD labelled protein co-elute. These the experiment point out together observe in the COS7 cell lysate ~ 500kD S kind may be the homotrimer of S albumen list aggressiveness.

The oligomeric state of S Δ spike protein also detects behind the COS7 cellular expression. Shown in Figure 81, when not having the heating pyrolyze thing before SDS-PAGE and the western blot analysis, the restructuring S Δ albumen that exists in the cell lysate also detects (swimming lane 1) with ~ 500 kDa scope HMW forms. Yet the oligomerization efficient of S Δ albumen seems than total length S albumen much lower (＜10%) under same protein engram analysis condition in the born of the same parents. This ~ test of the heat sensitivity of 500kDa albumen shows S Δ oligomer than total length S oligomer thermally labile more, this during by 80 ℃ all ~ 500kDa kind＞90% is transformed into single aggressiveness Sd form is proved (swimming lane 2). Equally (Figure 82), most of secretion S Δ albumen find with single aggressiveness form, and ~ the 500kDa kind almost can not detect (only when the excessive application of sample of albumen can detect during for western blot analysis) (swimming lane 1). In the temperature that is higher than 80 ℃, all secretion S Δ albumen that detect are single aggressiveness (swimming lanes 2,3).

~ 500kDa protein glycosylation, the research deglycosylation is on the impact of its antibody combination. Recombinant C OS7 lysate is processed under non-Denaturing (as mentioned above) with PNGase F and is carried out western blot analysis. Shown in Figure 83, deglycosylation does not affect anti-histidine Mab antibody in conjunction with the S oligomer of processing (swimming lane 2,3). Yet it has affected the reactivity (swimming lane 6) with the rabbit anti-serum of antivenom purification Virus culture. Only DTT is when omitting from the SDS-PAGE sample, and this antiserum in conjunction with the S that obtains from virion, indicates it mainly to identify discontinuous conformation epitope in western blot analysis. This antiserum also shows high virucidin and tires. It is not in conjunction with deglycosylation, restructuring S, and prompting carbohydrate positive role is in S polypeptide oligomer more orderly, natural structure.

Difference between restructuring S and S Δ albumen is the terminal existence of C or does not have TM-and Cys-to enrich domain. The prediction of this difference when the cracking transfectional cell, total length S and membrane portions link and Sd in soluble fraction. Therefore, nSh or the cracking under hypotonic condition of nSh Δ TC transfectional cell, solvable kytoplasm partly separates (Figure 48) by centrifugal and insoluble membrane portions. Shown in Figure 84, the S albumen (swimming lane 4) of discovery ~ 500kDa and 180/170kDa kind in the membrane portions (DF), but can not detect (swimming lane 3) in solvable kytoplasm part (AF). Yet, in 2 parts, all find the brachymemma S Δ albumen (swimming lane 5,6) of single aggressiveness kind (gp170). This shows that S albumen is anchored to cell membrane and needs the terminal TM-of C and Cys-to enrich domain.

Celluar localization by S and S Δ albumen in the indirect immunofluorescence microscopy Analysis for CO S7 cell. After the transfection 48 hours, cell was directly fixing with 2% paraformaldehyde, was used for the washing agent of cell surface dyeing, or processed with washing agent, then was for the Cytofix/Cytoperm solution that dyes in the born of the same parents. Put together the antibody staining fixed cell with the anti-SARS serum of rabbit (2BE) and FITC subsequently. The nSh transfectional cell shows S albumen transforming focus, indication Golgi localization (Figure 85 A), and nSh Δ TC transfectional cell shows S Δ albumen and is uniformly distributed in kytoplasm, indication ER location (Figure 85 B). Although in the on-fixed cell, also observe the lip-deep complete S albumen of transfectional cell (Figure 85 D), can not detect S Δ (Figure 85 E) at cell surface. These results indicate TM-and Cys-to enrich the effect that domain is brought into play in the plasma membrane at grappling S albumen. Although the TM district may act on the film grappling separately, the latent effect in the abundant district of Cys-still has to be determined.

Therefore, SARS recombinant full-lenght S albumen is the glycoprotein that N connects, and the estimation molecular weight is 170-180,000kDa. Produce the big or small polypeptide of expection with PNGase F deglycosylation, be used for non-cutting, coded polypeptide (140kDa). Comprise instantaneous in COS7,293, BHK21 and the Huh7 clone and stably express total length SARS-CoV S gene constant generation S albumen doublet (gp170/180) at multiple mammalian cell, detected such as western blot analysis. Pulse-chase analysis transfectional cell proof SARS-CoV S albumen synthesizes Endo H sensitiveness gp170 kind at first, Endo H resistance gp180 form then progressively occurs, and this may be owing to add complex carbohydrate and golgiosome.

Recombinant s protein is not secreted in the cell culture medium, unless contain terminal 60 amino acid deletions of C that TM district and Cys-enrich tail.

The quaternary structure of research total length recombinant s protein is used crosslinking Treatment, thermal denaturation and size fractionation compartment analysis. Result data exists consistent with recombinant s protein conduct ~ 500kDa homotrimer. Similar analysis obtains also to produce identical result from the S of virion. Other coating RNA virus was reported this tripolymer structure: the G albumen of the hemagglutinin HA of influenza virus, the E1-E2 heterodimer of Alphavirus and vesicular stomatitis virus. Under reducing condition, hatch and show that SARS-CoV S tripolymer structure is non-covalent and link and very stable. The S oligomer that exists in the cell lysate shows that anti-10mM DTT reduction, 1%SDS washing agent are processed and high thermal denaturation to 60 ℃. Hatch＞80 ℃ temperature and to cause the trimerization complex to dissociate, by tripolymer reduce and follow simultaneously single aggressiveness band increase proved. Temperature-induced gp170 (the single aggressiveness form of ER) and the complex glycosylation gp180 (golgiosome list aggressiveness form) that high mannose occurs, the prompting tripolymerization can occur before the golgiosome in the middle of single aggressiveness spike protein is transported to. This is consistent with other report about TGEV, influenza virus HA, vesicular stomatitis virus G albumen. For these albumen, the report tripolymer occurs before composite oligosaccharide adds golgiosome.

Find the terminal clipped form of C of the S of oligomerization and single aggressiveness form in cell lysate, frequency is respectively 10% and 90%. Discovery is secreted into the abundant glycosylation of truncated protein in the culture medium, and nearly all is single aggressiveness form. We infer that terminal 60 amino acid of the C of S glycoprotein comprise affects the film of tripolymer efficient anchorage zone. In S albumen tripolymer, may need the C end region to provide more stability force with three chain coiled coil structures in initial sequence of events and the S2 stem structure territory, as observing in the influenza virus HA oligomer.

Embodiment 24: be used for the Chinese hamster ovary celI that spike protein is expressed

The Chinese hamster ovary celI system of preparation stably express total length or brachymemma SARS-CoV spike protein. Obtain some stable transfected CHO cell lines, Figure 73 shows the Western blotting data from one group of representative clone.

Embodiment 25: at expression in escherichia coli

All SARS-CoV ORFs (Figure 17, table 10) are cloned in the pET carrier and are the terminal His mark of C fusion at expression in escherichia coli. Albumen less than 16KD also is expressed as terminal GST (glutathione S-transferase) fusion of N, uses the pET carrier.

Nsp1 and Nsp2 are 2 kinds of SARS-CoV albumen with proteolytic activity, because the toxicity in the Escherichia coli, they are not expressed as full-length proteins. Each gene changes in varing proportions the clone, be used for separately the catalytic residue of gained recombinant protein (for Nsp1, Cys833/His994; For Nsp2, His41/Cys145), the gained recombinant protein is: from AY310120 nucleotides 2719-5214; Nsp1B from nucleotides 5218-7371; Nsp1C from nucleotides 7372-9984; Nsp2A from nucleotides 9985-10416; Nsp2B from nucleotides 10476-10902.

Nsp9 (SEQ ID NO:9775) is divided into 2 parts: from the Nsp9A of nucleotides 13371-14756; Nsp9B from nucleotides 14757-16166.

Matrix (M), ORF3 and ORF7 comprise respectively 3,2 and 1 membrane spaning domains. These protein expressions are disappearance albumen, get rid of initial 100 amino acid (M and ORF3) or comprise front 18 amino acid (ORF7) of hydrophobic region.

Cloned sequence is shown in table 26.

2 step strategies are used for the amplification cloned sequence. In the 1st step, amplification contains the dna fragmentation that surpasses 1 gene or term single gene, uses the cDNA that checked order as template. 11 cDNA sequences increase: (1) fragment, and called after amplC1 comprises the gene of encoding proteins E, albumen M, orf 7-8-9-10; (2) fragment, called after amplC2 comprises the gene of coding orf 3-4; (3) fragment, called after amplC5 comprises the gene of encoding proteins Nsp12 and Nsp13; (4) Nsp11 gene; (5) P28 and P65 gene; (6) Nsp1B and Nsp1C Gene Partial; (7) fragment, called after amplC9 comprises the gene of encoding proteins Nsp2 and Nsp3; (8) fragment, called after amplNsp4-7 comprises encoding proteins Nsp4, Nsp5, Nsp6, Nsp7 and the gene of the Nsp9A Gene Partial that is used for increasing; (9) Nsp 9B Gene Partial and Nsp10 gene; (10) fragment, called after amplCO comprises the gene of encoding proteins Orf11, nucleocapsid protein (N) and Orf12; (11) Nsp1A Gene Partial. The 1st step the primer provides in table 27:

In the 2nd step, the amplification term single gene uses dna fragmentation from the 1st amplification step as template. Primer is shown in table 28.

In can be observed the albumen of expression, albumen or in inclusion body (insoluble) or be in soluble form. Carry out purifying at suitable material. Table 29 shows that SARS-CoV ORFs expresses the molecular weight of fragment, and whether they clone (+or-), whether observes the albumen form that cloned sequence is expressed (+or-) and selected to be used for purifying.

When albumen is solvable His marked product, rule single bacterium colony and on LB/Amp (100 μ g/ml) agar plate 37 ℃ of overnight growth. Come dull and stereotyped since then separation bacterium colony to be inoculated in 20ml LB/Amp (the 100 μ g/ml) fluid nutrient medium, 37 ℃ of shaken overnight growths. Overnight culture is diluted in 1.0L LB/Amp (the 100 μ g/ml) fluid nutrient medium at 1: 30, grows until OD550nm reaches 0.6-0.8 in optimum temperature (30 or 37 ℃). Induce expression of recombinant proteins and culture to hatch again 3 hours by adding IPTG (final concentration 1.0mM). By 4 ℃ of centrifugal 15 minutes collection bacteriums of 8000 * g. Bacterial precipitation is resuspended in 10ml cold buffer liquid A (300mM NaCl, 50mM phosphate buffer, 10mM imidazoles, pH 8.0). By ultrasonic degradation (French press) ruptured cell on ice, use Branson ultrasonic degradation device 450,40W 30 seconds 4 times, and centrifugal 30 minutes with 4 ℃ of 13000 * g. Supernatant mixes 150 μ l Ni²⁺-resin (front is crossed with the buffer A balance) and incubated at room, gentle agitation 30 minutes. Resin is that height flows chelating agarose (Chelating Sepharose Fast Flow) (Pharmacia), according to manufacturer's explanation preparation. Goods centrifugal 5 minutes of 4 ℃ of 700 * g, are abandoned supernatant in batches. Resin is washed 2 times (in batches) 10 minutes with the 10ml buffer A, is resuspended in the 1.0ml buffer A and is loaded onto disposable column. Resin is washed with 4 ℃ buffer A continuously, until the OD280nm that flows through reaches 0.02-0.01. Resin further uses cold buffer liquid B (300mM NaCl, 50mM phosphate buffer, 20mM imidazoles, pH 8.0) to wash, until the OD280nm that flows through reaches 0.02-0.01. Take off buffer solution C (300mM NaCl, 50mM phosphate buffer, 250mM imidazoles, pH 8.0) wash-out His fusion by adding 700 μ l cold wash, collect each several part until OD_280nmIndication obtains all recombinant proteins. Each wash-out part of 20 μ l five equilibriums is analyzed by SDS-PAGE. Measure the estimation protein concentration with Bradford.

When albumen is insoluble product, the following purifying of inclusion body: 4 ℃ of homogenize cells (5g weight in wet base) in 25ml 0.1M Tris HCl pH 7,1mM EDTA, use ultraturrax (10000rpm); Add the every gram cell of 1.5mg lysozyme; Hatched 30 minutes with the of short duration mixing of ultraturrax and 4 ℃; Come ruptured cell with ultrasonic degradation or high pressure homogenize; Be dna digestion, add MgCl₂To the 3mM final concentration and add DNase to the 10ug/ml final concentration, to hatch 30 minutes for 25 ℃, 7.0,4 ℃ of 60mM EDTA, 6%Triton x-100, the 1.5M NaClpH of adding 0.5 volume hatched 30 minutes in the solution; 4 ° of 31000g are centrifugal 10 minutes; Precipitation is resuspended in 40ml 0.1M Tris HClpH 7.0,20mM EDTA, uses ultraturrax; 4 ° of 31000g are centrifugal 10 minutes; The IB precipitation is stored in-20 ℃.

Expression of results is shown in Figure 86 to 105. The example of purity and output provides in table 30.

Embodiment 26: keep crucial epitope at brachymemma furcella antigen

Test has the active human monoclonal antibodies of neutralization and the reactivity of purifying brachymemma spike protein in ELISA measures. Briefly, the clipped form spike protein (100 μ l/ hole) of the coated 1 μ g/ml concentration of elisa plate, 4 ℃ of night incubation plates. The flushing plate, sealing non-specific binding site adds the antibody of different dilutions and plate subsequently incubated at room 1 hour. Hatch when finishing, the flushing plate also detects binding antibody, uses anti-human IgG and the suitable substrate of puting together horseradish peroxidase (HRP). The good density of each Kongzui is recorded as 405nm, uses the ELISA reader. Data are shown in Figure 69, and clear proof is by preserving with epitope in the mAb identification and being exposed on the restructuring brachymemma spike protein.

Embodiment 27: different furcella vaccines

The brachymemma spike protein of purifying is used for immune mouse, and the antibody that anti-brachymemma spike protein is induced is measured by ELISA in conjunction with level and determined. Briefly, 1 group of 10 mouse is immune with 3 μ g brachymemma spike proteins, and MF59 is as adjuvant, in the immunity of 0,4 and 8 weekly intervals. From these animals, collect blood serum sample, in ELISA measures, determine the antibody that the brachymemma spike protein is induced. Other 1 group of 8 mouse are with 75 μ g dna immunizations, and the clipped form spike protein on the dna encoding PLG particle in the immunity of 0,4 and 13 weekly intervals, is collected serum and as above analyzed anti-furcella antibody.

The binding antibody of inducing in each group distributes to paint and makes geometric mean titer (GMT). Also compare with the DNA vaccine of PLG microparticle formulation transmission with expressing brachymemma furcella antigen, the brachymemma spike protein of purifying obviously more effective powerful antibody of inducing reacts. Further compare the antibody response that deactivation BPL-SARS-CoV (having demonstrated protectiveness) induces in the same mouse strain, show the antibody response size in same range as (Figure 70) that purifying brachymemma spike protein and inactivated virus vaccine are induced.

Also assessment restructuring brachymemma spike protein or express identical furcella antigen antibody that DNA induces in and potential. 2 groups of gained GMT values are shown in Figure 71. According to these data, the as if more remarkable neutralizing antibody reaction of effectively inducing anti-SARS-CoV furcella of purifying protein. The neutralization of usually being induced by purifying brachymemma spike protein in addition, is tired to tire with the vaccine-induced neutralization of deactivation SARS-CoV and is compared.

Figure 72 shows antibody in conjunction with the comparison of level (ELISA, X-axis), and tire (Y-axis) that neutralize arranged. Generally, in conjunction with and neutralizing antibody between good correlation is arranged. The bottom left combination shows the ratio with 2 weeks after the 3rd immunity of dna vaccination; The combination of top the right shows the ratio with 2 weeks after the 2nd immunity of protein vaccine. 2 kinds of vaccine forms all show constant correlation.

In further testing, the researching DNA vaccine causes immunoreactive ability in mouse. Mouse is with the immunity of pCMV-nSdTC plasmid, and plasmid is not or the PLG particulate arranged. Be used as subsequently 293 cells, cell transfecting total length or the brachymemma furcella of the anti-cultivation of dyeing antibody from the serum of mouse. Centrifuge cell and cracking precipitation before the test. Test antibody, cleer and peaceful anti-cell lysate on the anti-culture. Shown in Figure 112, mice serum can detect spike protein in the cell conditioned medium of the cell lysate of expressing the total length furcella and expression brachymemma spike protein. The result can compare with the dyeing of observing with rabbit anteserum, and rabbit anteserum is to obtain after with complete killed vaccine immunity. Can use the dna vaccination inoculation to induce anti-furcella antibody.

Expression cassette among the embodiment 28:pCMV

The sequence of plasmid pCMVKm2 provides with SEQ ID NO:9923. Coding total length form (pCMVKm2 SARS furcella nS; SEQ ID NO:9921) or Δ TC form (pCMVKm2SARS furcella nS Δ TC; SEQ ID NO:9922) gene of spike protein inserts this underlying carrier.

Mouse is immune with the similar substrates of these carriers and coding N, M or E albumen. The carrier that also prepares the coding same protein but have optimal codon to select. The optimization codon is used for expressing from the effective people of FRA sequence (GenBank:AY310120) beginning. Optimal sequence is: N (SEQ ID NO:9924); M (SEQ ID NO:9925); E (SEQ ID NO:9926).

After using, in all situations, can detect protein expression by immunofluorescence. For example, Figure 106 shows immunofluorescence (with anti-SARS rabbit anteserum) result behind the carrier use coding optimal N antigen, shows high level expression. The mouse of accepting independent control vector does not show fluorescence.

The immunofluorescence of the more natural M sequence of Figure 107 (107A) or codon optimization M sequence (107B) (with the anti-M antibody of Abgent). The more natural E sequence of Figure 108 (108A)) or the immunofluorescence of codon optimization E sequence (108B) (with the anti-E antibody of Abgent) similarly.

Immunity 4 groups of mouse (8 every group) are used: (1) SARS nS furcella, nSdTC brachymemma furcella and N albumen; (2) pCMV-SARS-nSdTC:DNA+DNA-PLG is in 0,4 and 13 weeks; (3) CMV-nS:DNA+DNA-PLG+VEE/SIN Rep is in 0,4 and 9 weeks; (4) VEE/SIN Rep-SARS-nS, 3 times, in 0,4 and 13 weeks. All identify SARS nS and nSdTC albumen from the serum of all groups, show that also viral combination and neutralization are active.

Embodiment 29: the spike protein cutting

Be the effect of research protease cutting to the SARS-CoV spike protein, albumen is expressed in Escherichia coli in a variety of forms, comprising: (1) total length S1-S2; (2) independent S1; (3) seven HR1 and (4) seven HR2. The albumen of expressing is used for cultivating immunize rabbit serum, and serum is used for presenting the Western blotting of Vero cell subsequently, cell infection or do not infect SARS-CoV.

Figure 109 shows Western blotting, uses the antibody of dilution in 1: 10000, and the anti-S1 domain of antibody or uncut S1-S2 domain are cultivated. Figure 110 shows Western blotting, uses the antibody of dilution in 1: 10000, and anti-each the 4 kinds of albumen of antibody are cultivated. The difference of antigen reactivity is obvious.

Figure 111 shows the class likelihood data. 4 swimming lanes of each serologic test, these 4 swimming lanes from left to right are: (a) serum of dilution in 1: 500, SARS-CoV-infection cell; (b) serum of dilution in 1: 500, non-infected cell; (c) serum of dilution in 1: 2500, the SARS-CoV-infection cell; (d) serum of dilution in 1: 2500, non-infected cell. Again, the difference of antigen reactivity is obvious.

Figure 109-111 shows in the infected Vero cell and has multiple spike protein form, size be about 75kDa, 90kDa, 180kDa and＞250kDa. Cutting (small part) spike protein, cutting in born of the same parents or after particle discharges.

If the enzyme of mouse hepatitis coronavirus spike protein cutting is suppressed, then cell-Fusion of Cells (plasomidum form) is also suppressed, but virus-cell be not subjected to suppress (de Haan etc. (2004) J Virol). In SARS infected patient lung, observe plasomidum in the body, but in the Vero of SARS-CoV cell culture, do not find. Therefore, suppress the spike protein cutting and can be used for preventing Syncytium formation and related diseases Neo-Confucianism, even not blocking-up of viral infection.

Embodiment 30: purifying SRAS protease

Cell grows into mid-log phase at 37 ℃ and also induces with the 0.2%L-arabinose. Centrifugal collecting cell, cell is resuspended in lysis buffer (LB), and buffer solution contains 20mM Tris pH 7.5,500mM NaCl, 5% glycerine V/V, 0.05%Triton X-100,5mM ME, 5mM imidazoles and adequate proteins enzyme inhibitor (-) EDTA. Benzonase joins in the lysate of final concentration 50U/ml. Subsequently, come cell lysis by 2 times through precooling microjet homogenizer. Clarify lysate with 44,000 * g high speed centrifugation. The lysate of clarification is applied to ready Pharmacia chelating FF post, and the cornice electricity has nickelous sulfate. After using lysate, post is washed with the LB of 5 column volumes, and then the LB with 5 column volumes interpolation 45mM imidazoles washes. Subsequently with the LB wash-out post that adds the 250mM imidazoles. The purity of separating SARS protease is 50%. Collection contains the part of protease, adjusts to 5mM EDTA, then is applied to Superdex 200 solvent resistant columns, and post is the balance mistake in 20mM Tris pH 7.5,150mM NaCl, 5%V/V glycerine, 0.05%Triton X-100 and 5mM DTT. The purity of separating SARS protease is 70%. Again, collect the part that contains protease, subsequently-80 ° preservation is until use. Determination of activity, mass spectrum and western blot analysis are used for really identifying albumen (Figure 133). Institute finishes in the precooling buffer solution in steps, keeps as far as possible more multiple products at 4 ℃.

The Western blotting of SARS protease purification part

Operation: briefly, protein concentration is take the 280nm absorptance as the basis, and coomassie dyeing gel is estimated purity. Albumen runs at the 4-20% gradient gel, goes to nitrocellulose. Subsequently, point is with the 3%BSA sealing, with the anti-7His detection of mouse IgG, then with the mouse IgG two anti-detections of puting together HRP. Point presents with ECL kit (Pharmacia Biotech). The results are shown in Figure 133, wherein A is application of sample 50,100 and the set of the fractionation column of 200ng target protein, and B is application of sample 50,100 and the fixing metal affinity column set of 200ng target protein.

Embodiment 31: the continuous fluorescence resonance energy shifts (FRER) enzymatic determination

The peptide that contains EDANS, fluorescence donor, DABCYL, fluorescence quenching (DABCYL-VNSTLQ SGLRK-EDANS) is synthetic by Syn.Pep. (Dublin, CA). Peptide comprises cleavage site Gln-Ser in the centre. Meyers etc. " proteolytic enzyme handbook and Barrett, the .Academic Press such as A, London, 1998,726-728. Dynamics is followed the tracks of the proteolytic activity of SARS protease, contains the formation level of the cleaved products of fluorescence donor SGLRK-EDANS by measurement, uses Hitachi fluorescence photometer (F-4500FL Spec.), is set to 340nm and excites and 490 nm emission wavelengths. 5L 5mM peptide stoste in DMSO solution adds in the reactant mixture, mixture contains 295 l of standard buffer solutions (75mM Tris-Hcl, 25mM NaOAc, 25mM Bis-Tris, 25mM glycine, 5mM EDTA and 1mM EDTA, pH 7.4) and 100ul buffer solution or 100ul 3.6uM protease and stores liquid. Followed the tracks of kinetic curve 6 minutes (reaction is linear, and the R2 value is 0.998 (Figure 134)). It may be the enzyme dependence that fluorescence forms (protein cleavage reaction), when the enzyme concentration triplication, and the also triplication of fluorescence that in 6 minutes frames, forms.

Should be appreciated that the present invention only is described by the mode of embodiment, can make amendment and still remain in invention scope and the spirit.

Table 1. United States Patent (USP) and disclosed international patent application

Publication number	Title	Date of publication
			US-3927216	Suppress 1,2 of virus infections, 4-triazole E-3-carboxylic acid amides (1,2,4-Triazol E-3-Carboxamides For Inhibiting Virus Infections)	12/16/1975
US-4010269	Antiviral quinazoline composition and using method thereof (Antiviral Quinazoline Compositions And Methods Of Use)	3/1/1977
			US-4065570	Antiviral 5-(benzylidene of replacement) caprolactam class (Antiviral 5-(Substituted Benzal) Hydantoins)	12/27/1977
US-4089965	Thiazolyl guanidines (Thiazolylphenylguanidines As Antirhinovirus Agents) as the rhinovirus agent	5/16/1978
			US-4122191	Rhinovirus agent (Antirhinovirus Agents)	10/24/1978
US-4192895	Rhinovirus agent (Antirhinovirus Agents)	3/11/1980
			US-4254144	Cyanophenyl (Substituted Benzonitriles Having Antiviral Activity) with replacement of antiviral activity	3/3/1981
US-4264617	Antiviral 5-(benzylidene of replacement) caprolactam class (Antiviral 5-(Substituted Benzal) Hydantoins)	4/28/1981
			US-4287188	Purine derivative (Purine Derivatives)	9/1/1981
US-4327088	Phosphono oxygen-or the acrylophenone of glycosyl glycosyl oxygen-replacement, composition and use thereof (Phosphonooxy-Or Glycosyloxy-Substituted Acrylophenones, Compositions And Uses Thereof)	4/27/1982
			US-4332820	Cyanophenyl (Substituted Benzonitriles Having Antiviral Activity) with replacement of antiviral activity	6/1/1982
US-4349568	Diphenyl ether (Sulfur-Substituted Diphenyl Ethers Having Antiviral Activity) with the sulphur of antiviral activity-replacement	9/14/1982
			US-4352792	3-alkoxyl brass antivirotic (3-Alkoxyflavone Antiviral Agents)	10/5/1982
US-4371537	Phenoxypyridines (Sulfur-Substituted Phenoxypyridines Having Antiviral Activity) with the sulphur of antiviral activity-replacement	2/1/1983
			US-4423053	2-amino-5-(O-sulphamide phenyl)-1 as antivirotic, 3,4-thiadiazoles derivative and preparation method thereof (Derivatives Of 2-Amino-5-(O-Sulphamidophenyl)-1,3,4-Thiadiazol As Antiviral Agents And A Process For The Preparation Thereof)	12/27/1983
US-4505929	Diphenyl ether (Sulfur-Substituted Diphenyl Ethers Having Antiviral Activity) with the sulphur of antiviral activity-replacement	3/19/1985
			US-4526897	Hypertension agents isoindoline-2-base-aminooimidazole quinoline and isoindoline-2-base-guanidine (Hypertensive Isoindolin-2-Yl-Aminoimidazolines And Isoindolin-2-Yl-Guanidines)	7/2/1985
US-4558134	Some have the phenoxy group-pyridine of antiviral activity-nitrile (Certain Phenoxy-Pyridine-Carbonitriles Having Antiviral Activity)	12/10/1985
			US-4629729	Give anti--virus activity 2-alkyl amino-4,6-dihalo-pyrimidine (Endowed With Anti-Viral Activity 2-Alkylamino-4,6-Dihalo Pyrimidines)	12/16/1986
US-4636492	Suppress virus protein active (Inhibition Of Viral Protease Activity By Peptide Halomethyl Ketones) with peptide halogenated methyl ketone	1/13/1987
			US-4652552	The tetrapeptide MIBK inhibitor of virus protein (Tetrapeptide Methyl Ketone Inhibitors Of Viral Proteases)	3/24/1987
US-4724233	(Therapeutical Application Of Phosphonylmethoxyalkyl Adenines) used in the treatment of phosphonium mesitoyl methoxy alkyl adenine	2/9/1988
			US-4738984	Rhinovirus agent (Antirhinovirus Agents)	4/19/1988
US-4847246	From the antiviral composition of firefly and using method thereof (Antiviral Compositions Derived From Fireflies And Their Methods Of Use)	7/11/1989
			US-4855283	New pharmaceutically active N-(2-aminoacyl amino-2-deoxidation-hexose-based)-acid amides,-carbamate and application thereof (Novel Pharmaceutically Active N-(2-Aminoacylamido-2-Deoxy-Hexosyl)-Amides ,-Carbamates And-Ureas)	8/8/1989
US-4885285	Phosphorus compound, their manufacture method and their purposes (Phosphorus Compounds, Processes For Their Manufacture, And Their Use)	12/5/1989
			US-4956351	The anti-viral pharmaceutical compositions (Antiviral Pharmaceutical Compositions Containing Cyclodextrins) that contains cyclodextrin	9/11/1990
US-5001125	Anti--virus activity pyridazine pyridazine (Anti-Virally Active Pyridazinamines)	3/19/1991
			US-5036072	Antivirotic (Antiviral Agent)	7/30/1991
US-5070090	The morpholinyl alkyl phenol ether of Antipicornaviral heterocycle-replacement (Antipicorpaviral Herterocyclic-Substituted Morpholinyl Alkylphenol Ethers)	12/3/1991
			US-5100893	Antipicornaviral pyridazine (Antipicomaviral Pyridazinamines)	3/31/1992
US-5112825	The mix pyridazine (Antirhinoviral Heteroamine-Substituted Pyridazines) of amine-replacement of rhinovirus	5/12/1992
			US-5157035	Antiviral activity pyridazine (Anti-Virally Active Pyridazinamines)	10/20/1992
US-5240694	The combination anti-viral of common cold and anti-medium treatment (Combined Antiviral And Antimediator Treatment Of Common Colds)	8/31/1993
			US-5242924	As the tetrazole radical of antivirotic-(phenoxy group and phenoxyalkyl)-piperidyl pyridazine (Tetrazolyl-(Phenoxy And Phenoxyalkyl)-Piperidinylpyridazines As Antiviral Agents)	9/7/1993
US-5278184	Be fit to the pyrroles for the treatment of rhinovirus infection and buffering derivative (the Synthetic Derivatives Of Pyrrole And Pyrrolidine of pyrrolidines	1/11/1994

	Suitable For The Therapy Of Infections Caused By Rhinoviruses)
			US-5364865	As the phenoxy group of antivirotic-and phenoxyalkyl-piperidines (Phenoxy-And Phenoxyalkyl-Piperidines As Antiviral Agents)	11/15/1994
US-5453433	Thiadiazole material and Antipicornaviral composition (Thiadiazoles And Antipicornaviral Compositions)	9/26/1995
			US-5492689	The combination of common cold suppresses viral anti-medium (COVAM) treatment (Combined Virustatic Antimediator (COVAM) Treatment Of Common Colds)	2/20/1996
US-5514679	Therapeutic phenoxyalkyl pyridazine and intermediate thereof (Therapeutic Phenoxyalklpyridazines And Intermediates Therefor)	5/7/1996
			US-5514692	Be used as the quinoline (Substituted Quinoline Derivatives Useful As Antipiconaviral Agents) of the replacement of Antipicornaviral agent	5/7/1996
US-5523312	Antipicornaviral agent (Antipicornaviral Agents)	6/4/1996
			US-5545653	Anti--virus compound (Anti-Viral Compounds)	8/13/1996
US-5552420	Therapeutic phenoxyalkyl pyrroles and phenoxyalkyl azine (Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines)	9/3/1996
			US-5567719	Thiadiazoles and and as the application (Thiadiazoles And Their Use As Antipicornaviral Agents) of Antipicornaviral agent	10/22/1995
US-5580897	Has 1 of antifungal activity, 2-dithia cyclohexadiene (1,2-Dithiins Having Antifungal Activity)	12/3/1996
			US-5618821	Therapeutic phenoxyalkyl heterocycles (Therapeutic Phenoxyalkylheterocycles)	4/8/1997
US-5618849	Orally active antiviral compound (Orally Active Antiviral Compounds)	4/8/1997
			US-5648354	Has 1 of antifungal activity, 2-dithia cyclohexadiene (1,2-Dithiins Having Antifungal Activity)	7/15/1997
US-5650419	Thiadiazoles and and as the application (Thiadiazoles And Their Use As Antipicornaviral Agents) of Antipicornaviral agent	7/22/1997
			US-5693661	Anti--virus compound (Anti-Viral Compounds)	12/2/1997
US-5721261	Therapeutic phenoxyalkyl pyrroles and phenoxyalkyl azine (Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines)	2/24/1998
			US-5725859	Has the therapeutic agent based on plant (Plant-Based Therapeutic Agent With Virustatic And Antiviral Effect) that suppresses virus and antivirus action	3/10/1998
US-5750527	Thiadiazoles and and as the application (Thiadiazoles And Their Use As Antipicornaviral Agents) of Antipicornaviral agent	5/12/1998
			US-5750551	The treatment of virology disease (Treatment For Viral Diseases)	5/12/1998
US-5762940	Inhibition or break virus or retroviral method and composition (Methods And Compositions For Inhibiting Or Destroying Viruses Or Retroviruses)	6/9/1998
			US-5763461	Therapeutic phenoxyalkyl heterocycle (Therapeutic Phenoxyalkylheterocycles)	6/9/1998
US-5821242	Anti--virus compound (Anti-Viral Compounds)	10/13/1998
			US-5821257	Thiadiazoles and and as the application (Thiadiazoles And Their Uses As Antipicornaviral Agents) of Antipicornaviral agent	10/13/1998
US-5821331	Anti--picornavirus agent (Anti-Picornaviral Agents)	10/13/1998
			US-5846986	Therapeutic phenoxyalkyl pyrroles and phenoxyalkyl azine (Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines)	12/8/1998
US-5856530	Antipicornaviral compounds and their use and preparation method (Antipicornaviral Compounds And Methods For Their Use And Preparation)	1/5/1999
			US-5891874	Anti--virus compound (Anti-Viral Compound)	4/6/1999
US-5962487	Antipicornaviral compounds and their use and preparation method (Antipicornaviral Compounds And Methods For Their Use And Preparation)	10/5/1999
			US-6004933	Cystatin (Cysteine Protease Inhibitors)	12/21/1999
US-6020371	Antipicornaviral compounds, the composition that contains them and their use and preparation method (Antipicornaviral Compounds Compositions Containing Them And Methods For Their Use)	2/1/2000
			US-6087374	Anti--virus compound (Anti-Viral Compounds)	7/11/2000
US-6114327	Anti--virus compound (Anti-Viral Compounds)	9/5/2000
			US-6117844	The method and composition (Method And Composition For Antiviral Therapy) that is used for antiviral therapy	9/12/2000
US-6194447	Two (benzimidazole) derivatives (Bis (Benzimidazole) Derivatives Serving As Potassium Blocking Agents) as the potassium blocking agent	2/27/2001
			US-6214799	Antipicornaviral compounds and their use and preparation method (Antipicornaviral Compounds And Methods For Their Use And Preparation)	4/10/2001
US-6277891	Nitric oxide suppresses rhinovirus infection (Nitric Oxide Inhibits Rhinovirus Infection)	8/21/2001
			US-6294186	The antimicrobial compositions (Antimicrobial Compositions Comprising A Benzoic Acid Analog And A Metal Salt) that contains benzoic acid analog and slaine	9/25/2001
US-6331554	Antipicornaviral compounds contains their composition and their use and preparation method (Antipicornaviral Compounds, Compositions Containing Them, And Methods For Their Use)	12/18/2001
			US-6358971	Anti--virus compound (Anti-Viral Compounds)	3/19/2002
US-6362166	Antipicornaviral compounds and their use and preparation method (Antipicornaviral Compounds And Methods For Their	3/26/2002

	Use And Preparation)
			US-6414004	Has the 5-aryl that antiviral activity 3-replaces-4-isoxazole nitrile (3-Substituted 5-Aryl-4-Isoxazolecarbonitriles Having Antiviral Activity)	7/2/2002
US-6420591	Carbamate and composition thereof, treat method (the Carbamates And Compositions Thereof of cancer, inflammation or virus infections with them, And Methods For Their Use For Treating Cancer, Inflammation, Or A Viral Infection)	7/16/2002
			US-6469018	Compound (Compounds)	10/22/2002
US-6498178	IMPDH enzyme inhibitor (Inhibitors Of IMPDH Enzyme)	12/24/2002
			US-6514997	Antipicornaviral compounds and composition, their medicinal application, and their synthetic (Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Their Synthesis)	2/4/2003
US-6525043	The use of ion channel modulator (Use Of Ion Channel Modulating Agents)	2/25/2003
			US-6531452	Antipicornaviral compounds and composition, their medicinal application, and their synthetic (Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Their Synthesis)	3/11/2003
US-6534489	Organic phosphorus compound and use thereof (Organophosphorus Compounds And The Use Thereof)	3/18/2003
			WO00/06529	As two ketone acids of AG14361-derivative (Diketoacid-Derivatives As Inhibitors Of Polynerases)	2/10/2000
WO00/25791	The pyrazine (Pyridin-4-Yl Or Pyrimidin-4-Yl Substituted Pyrazines) that pyridin-4-yl or pyrimidine-4-yl replace	5/11/2000
			WO00/27423	The method and composition (Methods And Compositions For Treating Common Cold Symptoms) for the treatment of common cold symptoms	5/18/2000
WO00/34308	Protein transduction guiding systems and using method thereof (Protein Transduction System And Methods Of Use Thereof)	6/15/2000
			WO00/39348	Identify the method and composition (Methods And Compositions For Identifying Protease Modulators) of protease adjusting	7/6/2000
WO00/40243	Novel compound (Novel Compounds)	7/13/2000
			WO00/50037	Nitrosylation and proton pump inhibitor nitrosyl radical, composition and using method thereof (Nitrosated And Nitrosylated Proton Pump Inhibitors, Compositions And Methods Of Use)	8/31/2000
WO00/56331	Impdh enzyme inhibitor (Inhibitors Of Impdh Enzyme)	9/28/2000
			WO00/56757	Immunomodulator steroids, the especially semihydrate of 16. α .-bromine epiandrosterones (Immunomodulatory Steroids, In Particular The Hemihydrate Of 16.Alpha.-Bromoepiandrosterone)	9/28/2000
WO00/66096	The new usage of Anti-epileptics and medicine (New Indication For Use Of Antiepileptic Agents And Medicines)	11/9/2000
			WO00/78746	Antivirotic (Antiviral Agents)	12/28/2000
WO01/00199	The compound with antiviral activity (Compounds Obtained From Salvia Species Having Antiviral Activity) available from Salvia	1/4/2001
			WO01/00585	Pyrazolidine alcoholic compound (Pyrazolidinol Compounds)	1/4/2001
WO01/02551	Virus-like particle, the preferably application in drug screening and functional genomics of their preparation and they (Virus Like Particles, Their Preparation And Their Use Preferably In Pharmaceutical Screening And Functional Genomics)	1/11/2001
			WO01/03681	The application (Use Of Flavones, Coumarins And Related Compounds To Treat Infections) in treatment is infected of flavonoids, Coumarins and related compound	1/18/2001
WO01/05396	Use chelating cobalt therapy or pre-preventing virus infection (Use Of Cobalt Chelates For Treating Or Preventing Virus Infection)	1/25/2001
			WO01/10894	Antipicornaviral compounds and composition, their medicinal application, and their synthetic (Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Their Synthesis)	2/15/2001
WO01/19322	Csaids is used for rhinovirus infection (Use Of Csaids In Rhinovirus Infection)	3/22/2001
			WO01/19822	Antivirotic (Antiviral Agents)	3/22/2001
W001/22920	Colon and colon cancer related polynucleotides and polypeptide (Colon And Colon Cancer Associated Polynucleotides And Polypeptides)	4/5/2001
			WO01/25188	New carbamate and urea (Novel Carbamates And Ureas)	4/12/2001
WO01/31016	The human cell factor Phc-1 and the Phc-2 (Processed Human Chemokines Phc-1 And Phc-2) that process	5/3/2001
			WO01/37837	3,4-dihydro-(1h)-quinazoline-2-ketone and they purposes (3,4-Dihydro-(1h)-Quinazolin-2-Ones And Their Use As Csbp/P38 Kinase Inhibitors) as the Csbp/P38 inhibitors of kinases	5/31/2001
WO01/38312	Quinazoline-2-the ketonic compound of 3,4-dihydro-(1h) is as Csbp/P38 inhibitors of kinases (3,4-Dihydro-(1h) Quinazolin-2-One Compounds As Csbp/P38 Kinase Inhibitors)	5/31/2001
			WO01/38313	Quinazoline-2-the ketonic compound of 3,4-dihydro-(1h) is as Csbp/P38 inhibitors of kinases (3,4-Dihydro-(1h) Quinazolin-2-One Compounds As Csbp/P38 Kinase Inhibitors)	5/31/2001
WO01/38314	Quinazoline-2-the ketonic compound of 3,4-dihydro-(1h) is as Csbp/P38 inhibitors of kinases (3,4-Dihydro-(1h) Quinazolin-2-One Compounds As Csbp/P38 Kinase Inhibitors)	5/31/2001
			WO01/40189	Antipicornaviral compounds and composition, their medicinal application, and their synthetic (Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Their Synthesis)	6/7/2001
WO01/49303	Multivalence electronically active composition and their method of manufacture and use thereof (Multivalent Electron Active Compositions And	7/12/2001

	Methods Of Making And Using Same)
			WO01/60393	With human immunodeficiency virus's selective destruction cell infection (Selective Destruction Of Cells Infected With Human Immunodeficiency Virus)	8/23/2001
WO01/62726	2-oxygen-1-pyrrolidin derivatives, their manufacture method and purposes (2-Oxo-1-Pyrrolidine Derivatives, Processes For Preparing Them And Their Uses)	8/30/2001
			WO01/79167	Antipicornaviral compounds and composition, their medicinal application, and their synthetic (Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Their Synthesis)	10/25/2001
WO01/90047	New Mmp-2/Mmp-9 inhibitor (Novel Mmp-2/Mmp-9 Inhibitors)	11/29/2001
			WO01/90129	Use based on compound prevention and the treatment of monose and polysaccharide and infect and Other diseases (Prophylactic And Therapeutic Treatment Of Infectious And Other Diseases With Mono-And Disaccharide-Based Compounds)	11/29/2001
WO01/92499	Nucleic acid molecules and the Ser/Thr kinases Akt interaction (Nucleic Acid Molecules Encoding A Protein Interacting With Ser/Thr Kinase Akt) of coding A albumen	12/6/2001
			WO01/93883	Therapeutic agent-Iii (Therapeutic Agents-Iii)	12/13/2001
WO01/93884	Therapeutic agent-I (Therapeutic Agents-I)	12/13/2001
			WO01/93885	Therapeutic agent-Ii (Therapeutic Agents-Ii)	12/13/2001
WO01/96297	Antipicornaviral compounds and composition, their medicinal application, and their synthetic (Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Their Synthesis)	12/20/2001
			WO02/04413	The chirality integrin is regulated son and using method (Chiral Integrin Modulators And Methods Of Use Thereof) thereof	1/17/2002
WO02/11743	The treatment of prostate cancer (Treatment Of Prostate Cancer)	2/14/2002
			WO02/12477	Strengthen copy (the Enhanced Replication Of Hcv Rna) of Hcv Rna	2/14/2002
WO02/14343	Compound (Immunosuppressive, Antiinflammatory And Analgetic Compounds) Immunosuppression, anti-inflammatory and pain relieving	2/21/2002
			WO02/24145	Antiviral substance (Antiviral Substances From Plant Cuticular And Epicuticular Material) from Plant cuticle and upper leather material	3/28/2002
WO02/28351	Recombinant paramyxovirus protein-binding protein (Recombinant Mucin Binding Proteins From Steptococcus Pneumoniae) from streptococcus pneumonia	4/11/2002
			WO02/30442	Treat the method (Method For Treating Cytokine Mediated Hepatic Injury) of cytokine mediated hepatic injury	4/18/2002
WO02/34771	Nucleic acid and protein (Nucleic Acids And Proteins From Streptococcus Groups A﹠B) from streptococcus group A and B	5/2/2002
			WO02/44737	The diagnosis and therapeutic combination and the method (Diagnostic And Therapeutic Compositions And Methods Related To G Protein-Coupled Receptor (Gpcr) Anaphylatoxin C3a Receptor) that relate to G albumen-coupled receptor (Gpcr) anaphylatoxin C3a receptor	6/6/2002
WO02/50045	Antivirotic (Antiviral Agents)	6/27/2002
			WO02/51413	Large ring resists-virus compound (Macrocyclic Anti-Viral Compounds)	7/4/2002
WO02/53138	Suppress the treatment (Treatment For Inhibiting Neoplastic Lesions) of knurl damage	7/11/2002
			WO02/57425	Nucleoside derivates (Nucleoside Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase) as RNA dependent form RNA viral polymerase inhibitors	7/25/2002
WO02/59083	New compound (Novel Compounds)	8/1/2002
			WO02/60875	Niacinamide diaryl derivatives (Nicotinamide Biaryl Derivatives Useful As Inhibitors Of Pde4 Isozymes) as the Pde4 isomerase inhibitors	8/8/2002
WO02/60898	As the thiazolyl of Pde4 isomerase inhibitors-oxazolyl-, pyrrole radicals-and imidazoles agent-acid amide derivatives (Thiazolyl-, Oxazolyl-, Pyrrolyl-, And Imidazolyl-Acid Amide Derivatives Useful As Inhibitors Of Pde4 Isozymes)	8/8/2002
			WO02/69903	Nucleosides, its preparation and as the application (Nucleosides, Preparation Thereof And Use As Inhibitors Of Rna Viral Polymerases) of RNA viral polymerase inhibitors	9/12/2002
WO02/72022	Tetracycline compound (Substituted Tetracycline Compounds As Antifungal Agents) as the replacement of antifungal agent	9/19/2002
			WO02/72031	Tetracycline compound (Substituted Tetracycline Compounds As Synergistic Antifungal Agents) as the replacement of working in coordination with antifungal agent	9/19/2002
WO02/76939	Cystatin (Cysteine Protease Inhibitors)	10/3/2002
			W002/77021	Streptococcus pneumoniae proteins and nucleic acid (Streptococcus Pneumoniae Proteins And Nucleic Acids)	10/3/2002
WO02/79401	New Rgs9 protein combination interaction and using method thereof (Novel Rgs9 Protein Binding Interactions And Methods Of Use Thereof)	10/10/2002
			WO02/82041	The new reagent based on peptide that manufacturing and use are used with the bispecific acceptor (Production And Use Of Novel Peptide-Based Agents For Use With Bi-Specific Antibodies)	10/17/2002
WO02/87465	The two target compositions of virus infections and cancer cell and method (Compositions And Methods Of Double-Targeting Virus	11/7/2002

	Infections And Cancer Cells)
			WO02/87500	The Prototoxophores of viral enzyme activation and treat virus infections (Viral Enzyme Activated Prototoxophores And Use Of Same To Treat Viral Infections) with them	11/7/2002
WO02/88091	Suppress ERC group virus's 2a cysteine proteinase (Inhibitors Of Human Rhinovirus 2a Cysteine Protease)	11/7/2002
			WO02/89832	By regulating the pharmaceutical composition (Pharmaceutical Compositions For Preventing Or Treating Th1 And Th2 Cell Related Diseases By Modulating The Th1/Th2 Ratio.) of the prevention of Th1/Th2 ratio or treatment Th1 and Th2 cell related diseases	11/14/2002
WO02/92779	Make tissue be rich in the method (Method For Enriching Tissues In Long Chain Polyunsaturated Fatty Acids) of long-chain polyunsaturated fatty acid	11/21/2002
			WO02/94185	Be used for carrying conjugate and the composition (Conjugates And Compositions For Cellular Delivery) of cell	11/28/2002
WO02/94868	Aureus protein and nucleic acid (Staphylococcus Aureus Proteins And Nucleic Acids)	11/28/2002
			WO02/96867	Be used for treating the kinases inhibitor (Inhibitors Of Protein Kinase For The Treatment Of Disease) of disease	12/5/2002
WO02/98424	Novel resistant to infection agent (Novel Anti-Infectives)	12/12/2002
			WO03/04489	The composition and the method (Compositions And Methods For Inhibiting Prenyltransferases) that suppress prenyltransferase	1/16/2003
WO03/08628	Enzymatic nucleic acid peptide conjugate (Enzymatic Nucleic Acid Peptide Conjugates)	1/30/2003
			WO03/15744	Chitin particulate and medical application thereof (Chitin Microparticles And Their Medical Uses)	2/27/2003
WO03/20222	Dioxolanes and oxa-mercaptan alkane derivatives are as the RNA viral polymerase inhibitors (Dioxolane And Oxathiolane Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase) that depends on RNA	3/13/2003
			WO03/20270	Oxadiazolyl-phenoxyalkyl isoxazole, its composition and they are used as the method (Oxadiazolyl-Phenoxyalkylisoxazoles, Compositions Thereof And Methods For Their Use As Anti-Picornaviral Agents) of anti--picornavirus agent	3/13/2003
WO03/20271	Oxadiazolyl-phenoxyalkyl isoxazole, its composition and they are used as the method (Oxadiazolyl-Phenoxyalkylisoxazoles, Compositions Thereof And Methods For Their Use As Anti-Picornaviral Agents) of anti--picornavirus agent	3/13/2003
			WO03/20712	Oxadiazolyl-phenoxyalkyl isoxazole, its composition and they are used as the method (Oxadiazolyl-Phenoxyalkylisoxazoles, Compositions Thereof And Methods For Their Use As Anti-Picornaviral Agents) of anti--picornavirus agent	3/13/2003
WO86/03412	The improvement (Improvements Relating To The Treatment Control And Prevention Of Rhinovirus Infections) of Control and prevention rhinovirus infection treatment	6/19/1986
			WO86/03971	Antivirotic (Antiviral Agents)	7/17/1986
WO88/09669	Avirulent microbes and application thereof (Avirulent Microbes And Uses Therefor)	12/15/1988
			WO92/03475	Enterovirus peptide (Enterovirus Peptides)	3/5/1992
WO92/22520	Orally active antiviral compound (Orally Active Antiviral Compounds)	12/23/1992
			WO92/22570	Picornavirus protease inhibitors (Inhibitors Of Picornavirus Proteases)	12/23/1992
WO94/00012	Nucleic acid and with their control viral pathogen method (Nucleic Acids And Methods Of Use Thereof For Controlling Viral Pathogens)	1/6/1994
			WO95/03821	Prosaposin and cell factor derived peptide (Prosaposin And Cytokine-Derived Peptides As Therapeutic Agents) as therapeutic agent	2/9/1995
WO95/09175	Ring-expansion nucleosides and nucleotides (Ring-Expanded Nucleosides And Nucleotides)	4/6/1995
			WO95/11992	Antiviral compound (Antiviral Compounds)	5/4/1995
WO95/31198	Thiadiazoles and and as the application (Thiadiazoles And Their Use As Antipicornaviral Agents) of Antipicornaviral agent	11/23/1995
			WO95/31438	Therapeutic phenoxyalkyl heterocycle (Therapeutic Phenoxyalkylheterocycles)	11/23/1995
WO95/31439	Therapeutic phenoxyalkyl pyridazine and intermediate thereof (Therapeutic Phenoxyalkylpyridazines And Intermediates Therefor)	11/23/1995
			WO95/31452	Therapeutic phenoxyalkyl pyrroles and phenoxyalkyl azine (Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines)	11/23/1995
WO95/34595	Antiviral arborescence (Antiviral Dendrimers)	12/21/1995
			WO95/35103	Prevent and/or treat pharmaceutical composition and the methods for the treatment of (A Pharmaceutical Composition For The Prevention And/Or Treatment Of Viral Infections And Optionally Inflammations As Well As A Method For The Treatment Thereof) of virus infections and optional inflammation	12/28/1995
WO96/05836	Method (Methods Of Treating Cold Symptoms Using Pentoxifylline) with PTX treatment cold symptoms	2/29/1996
			WO96/05854	Preparation contains the composition (Combination Preparation, Containing Cyclosporin A Or Fk506 Or Rapamycin And A Xanthine Derivative) of cyclosporin A or Fk506 or rapamycin and xanthine derivative	2/29/1996
WO96/09822	Antipicornaviral agent (Antipicornaviral Agents)	4/4/1996
			WO96/11211	The selective RNA translation (Selective Inhibition Of Internally Initiated Rna Translation) that suppresses to start from inside	4/18/1996

WO96/22689	Multicomponent RNA catalyst and application thereof (Multiple Component Rna Catalysts And Uses Thereof)	8/1/1996
			WO96/40641	As the sulphone amide derivative of assisting a ruler in governing a country instrumentality (Sulfonamide Derivatives As Cell Adhesion Modulators)	12/19/1996
WO97/08553	The protein of target-seeking gram-positive bacteria cell wall (Targeting Of Proteins To The Cell Wall Of Gram-Positive Bacteria)	3/6/1997
			WO97/34566	The electrophilic ketone (Electrophilie Ketones For The Treatment Of Herpesvirus Infections) for the treatment of herpesvirus infection	9/25/1997
WO97/41137	The application of anthocyanidin and anthocyanidin derivatives (Use Of Anthocyanidin And Anthocyanidin Derivatives)	11/6/1997
			WO97/43305	Picornavirus HRV 3CP inhibitor and their use and preparation method (Inhibitors Of Picomavirus 3c Proteases And Methods For Their Use And Preparation)	11/20/1997
WO97/47270	Novel antivirus compound (Novel Anti-Viral Compounds)	12/18/1997
			WO98/03572	Linear antiviral polymeric (Antiviral Linear Polymers)	1/29/1998
WO98/07745	Composition and method (Compositions And Methods For Treating Infections Using Analogues Of Indolicidin) with the infection of Indolicidin analogue treatment	2/26/1998
			WO98/11778	The antimicrobial therapy (Antimicrobial Treatment For Herpes Simplex Virus And Other Infectious Diseases) of herpes simplex virus and other infectious diseases	3/26/1998
WO98/22495	Anti-kassinin kinin compound and application thereof (Antikinin Compounds And Uses Thereof)	5/28/1998
			WO98/31363	Anti--virus compound (Anti-Viral Compounds)	7/23/1998
WO98/31374	The method (Method Of Treating Rhinoviral Infections) for the treatment of rhinovirus infection	7/23/1998
			WO98/32427	Infect (Therapeutic Treatment And Prevention Of Infections With A Bioactive Material Encapsulated Within A Biodegradable-Biocompatible Polymeric Matrix) with the bioactivator treatment and the prevention that are wrapped in biodegradable-biocompatibility polymeric matrices	7/30/1998
WO98/34601	The method (Method For Inhibiting Intracellular Viral Replication) that suppresses born of the same parents' viral replication in	8/13/1998
			WO98/42188	Antimicrobial prevention and the treatment (Antimicrobial Prevention And Treatment Of Human Immunedeficiency Virus And Other Infectious Diseases) of human immune deficiency type virus and other infectious diseases	10/1/1998
WO98/43950	Antipicornaviral compounds contains their composition and their using method (Antipicornaviral Compouds, Compositions Containing Them, And Methods For Their Use)	10/8/1998
			WO98/49190	Replace De oxadiazole cystatin (Substituted Oxadiazole Cysteine Protease Inhibitors)	11/5/1998
WO98/55120	Anti--virus compound (Anti-Viral Compounds)	12/10/1998
			WO99/30699	Cysteine proteinase instrumentality (Anti-Viral Compounds)	6/24/1999
WO99/31122	Antipicornaviral compounds and their use and preparation method (Antipicornaviral Compounds And Methods For Their Use And Preparation)	6/24/1999
			WO99/54317	Cystatin (Cysteine Protease Inhibitors)	10/28/1999
WO99/55663	Impdh enzyme inhibitor (Inhibitors Of Impdh Enzyme)	11/4/1999
			WO99/57135	Antipicornaviral compounds, their preparation and use (Antipicornaviral Compounds, Their Preparation And Use)	11/11/1999
WO99/59587	Anti--virus compound (Anti-Viral Compounds)	11/25/1999
			WO99/61437	The imidazolium compounds (Novel 2-Alkyl Substituted Imidazole Compounds) that novel 2-alkyl replaces	12/2/1999

Table 2. United States Patent (USP) and disclosed international patent application

Publication number	Title	Date of publication
			WO02/69903	Nucleosides, its preparation and as the application (Nucleosides, Preparation Thereof And Use As Inhibitors Of Rna Viral Polymerases) of RNA viral polymerase inhibitors	9/12/2002
WO02/48116	Hepatitis C virus Ns3 protease inhibitors (Inhibitors Of Hepatitis C Virus Ns3 Protease)	6/20/2002
			WO02/48157	Imidazolone and related derivatives (Imidazolidinones And Their Related Derivatives As Hepatitis C Virus Ns3 Protease Inhibitors) thereof as hepatitis C virus Ns3 protease inhibitors	6/20/2002
WO02/61048	The replication in vitro system (In Vitro System For Replication Of Rna-Dependent Rna Polymerase (Rdrp) Viruses) of RNA-directed RNA polymerase (Rdrp) virus	8/8/2002
			WO03/02518	Novel 2 as antivirotic, 4-difluorobenzamide derivative (Novel 2,4-Difluorobenzamide Derivatives As Antiviral Agents)	1/9/2003
WO02/79187	As the methoxyl group of antivirotic-1,3,5-triazines derivative (Methoxy-1,3,5-Triazine Derivatives As Antiviral Agents)	10/10/2002
			WO01/78648	6-methylnicotinamide derivative (6-Methylnicotinamide Derivatives As Antiviral Agents) as antivirotic	10/25/2001
WO01/12214	Mycophenolate is combined (MYCOPHENOLATE MOFETIL IN ASSOCIATION WITH PEG-IFN-.Alpha.) with PEG-IFN-α	2/22/2001
			WO02/100415	The nucleosides of 4 '-replacement (4 '-Substituted Nucleosides)	12/19/2002
WO02/18404	Nucleoside derivates (Nucleoside Derivatives)	3/7/2002

WO02/94289	Antiviral nucleoside derivatives (Antiviral Nucleoside Derivatives)	11/28/2002
			WO96/39500	Hepatitis C virus specific oligonucleotide (Oligonucleotides Specific For Hepatitis C Virus)	12/12/1996
WO03/00713	The nucleoside compound of HCV (Nucleoside Compounds In Hcv)	1/3/2003
			WO01/60381	Encircle the nucleoside analog (Nucleoside Analogs With Carboxamidine-Modified Bicyclic Base) of bases with two of carbonamidine modification	8/23/2001
WO02/03997	Pyrido [2,3-D] pyrimidine and pyrimido [4,5-D] pyrimidine nucleoside (Pyrido[2,3-D] Pyrimidine And Pyrimido[4,5-D] Pyrimidine Nucleosides)	1/17/2002
			WO97/26883	In the T-thymus dependent cells that activates, regulate Th1/Th2 cytokine-expressing (Modulation Of Th1/Th2 Cytokine Expression By Ribavirin3 And Ribavirin3 Analogs In Activated T-Lymphocytes) by Ribavirin 3 and Ribavirin 3 analogs	7/31/1997
WO03/26589	With 4 '-method and composition of the nucleosides treatment hepatitis C virus modified (Methods And Compositions For Treating Hepatitis C Virus Using 4 '-Modified Nucleosides)	4/3/2003
			WO03/26675	With 4 '-the nucleosides treatment flavivirus of modifying and the method and composition of pestivirus (Methods And Compositions For Treating Flaviviruses And Pestiviruses Using 4 '-Modified Nucleoside)	4/3/2003
WO97/30067	Sugar-modified gapped oligonucleotides (Sugar-Modified Gapped Oligonucleotides)	8/21/1997
			WO01/47883	Fused ring compound and as the application (Fused-Ring Compounds And Use Thereof As Drugs) of medicine	7/5/2001
WO03/00254	Fused ring compound and as the application (Fused Cyclic Compounds And Medicinal Use Thereof) of medicine	1/3/2003
			WO02/100354	Pyrrolo-[2,3-D] pyrimidine nucleoside analoys (Pyrrolo[2,3-D] Pyrimidine Nucleoside Analogs)	12/19/2002
WO01/55111	Biaryl compound, its preparation and therapeutical uses (Biaryl Compounds, Their Preparation And Their Use In Therapy)	8/2/2001
			WO01/16149	2-azapurine compound and uses thereof (2-Azapurine Compounds And Their Use)	3/8/2001
WO01/85770	Whistle virus Ii (Sentinel Virus Ii)	11/15/2001
			WO02/12263	Contain purine-2, the pyrazolo [3 of 6-diamines, 4-D] nucleic acid binding compounds and application (the Nucleic Acid Binding Compounds Containing Pyrazolo[3 thereof of pyrimidine analogue, 4-D] Pyrimidine Analogues Of Purin-2,6-Diamine And Their Uses)	2/14/2002
JP 2001-247550 A2	Fused ring compound and medical application thereof (Condensed Ring Compound And Its Medicinal Use)	9/11/2001
			6210675	PT-NANB hepatitis polypeptide (PT-NANB Hepatitis Polypeptides)	4/3/2001
6451991	Sugar-modified gapped oligonucleotides (Sugar-Modified Gapped Oligonucleotides)	9/17/2002
			5830455	With alpha-interferon and the method (Method Of Treatment Using A Therapeutic Combination Of α-Interferon And Free Radical Scavengers) for the treatment of without the therapeutic composition of radical scavengers	11/3/1998
5908621	Polyethyleneglycol modified interferon therapy (Polyethylene Glycol Modified Interferon Therapy)	6/1/1999
			5990276	The synthetic inhibitor of hepatitis C virus NS 3 protease (Synthetic Inhibitors Of Hepatitis C Virus NS3 Prptease)	11/23/1999
6172046	Eliminate the therapeutic alliance (Combination Therapy For Eradicating Detectable HCV-RNA In Patients Having Chronic Hepatitis C Infection) that can detect HCV-RNA among the chronic hepatitis C infection patient	1/9/2001
			6177074	Polyethyleneglycol modified interferon therapy (Polyethylene Glycol Modified Interferon Therapy)	1/23/2001
6326137	The chimeric pestivirus of hepatitis C virus protease dependent form (Hepatitis C Virus Protease-Dependent Chimeric Pestivirus)	12/4/2001
			6434489	Hepatitis C virus NS 5 B polymerase conjugate and method for crystallising thereof (Compositions Of Hepatitis C Virus NS5B Polymerase And Methods For Crystallizing Same)	8/13/2002
6461605	The low dosage cell factor is inculcated treatment (Continuous Low-Dose Cytokine Infusion Therapy) continuously	10/8/2002
			6472373	Eliminate the therapeutic alliance (Combination Therapy For Eradicating Detectable HCV-RNA In Antiviral Treatment Naive Patients Having Chronic Hepatitis C Infection) that can detect HCV-RNA among the chronic hepatitis C infection patient	10/29/2002
6524570	Polyethyleneglycol modified interferon therapy (Polyethylene Glycol Modified Interferon Therapy)	2/25/2003
			WO00/37097	Ribavirin-interferon alpha-induced HCV therapeutic alliance (Ribavirin-Interferon Alfa Induction Hcv Combination Therapy)	6/29/2000
WO00/37110	Ribavirin-PEG-IFN α induces HCV therapeutic alliance (Ribavirin-Pegylated Interferon Alfa Induction Hev Combination Therapy)	6/29/2000
			WO00/62799	The HCV therapeutic alliance contains the Ribavirin (Hcv Combination Therapy, Containing Ribavirin In Association With Antioxidants) of being combined with antioxidant	10/26/2000
WO01/58929	Treat the azepine peptide class (Azapeptides Useful In The Treatment Of Hepatitis C) of the third liver	8/16/2001
			WO02/32414	Ribavirin-PEG-IFN α induces HCV therapeutic alliance (Ribavirin-Pegylated Interferon Alfa Hcv Combination Therapy)	4/25/2002
WO03/24461	HCV therapeutic alliance (Hcv Combination Therapy)	3/27/2003
			WO93/20835	Treat hepatitis (Treatment Of Hepatitis With Gm-Csf) with Gm-Csf	10/28/1993
WO96/36702	The active hepatitis C virus protease (Soluble, Active Hepatitis C Virus Protease) of solubility	11/21/1996
			WO97/16204	The low dosage cell factor is inculcated treatment (Continuous Low-Dose Cytokine Infusion Therapy) continuously	5/9/1997
WO97/43310	Hepatitis C virus Ns3 protease synthetic inhibitor (Synthetic Inhibitors Of Hepatitis C Virus Ns3 Protease)	11/20/1997

WO98/48840	The polyethylene glycol-interferon alpha conjugate (Polyethylene Glycol-Interferon Alpha Conjugates For Therapy Of Infection) that treatment is infected	11/5/1998
			WO99/15194	Eliminate the therapeutic alliance (Combination Therapy For Eradicating Detectable Hcv-Rna In Patients Having Chronic Hepatitis C Infection) that can detect HCV-RNA among the chronic hepatitis C infection patient	4/1/1999
WO99/59621	Ribavirin and interferon-' alpha ' therapeutic alliance are used for first chronic hepatitis C infection patient's antiviral therapy (Combination Therapy Comprising Ribavirin And Interferon Alpha In Antiviral Treatment Naive Patients Having G Chronic Hepatitis C Infection)	11/25/1999
			WO02/100846	The Compounds and methods for (Compounds And Methods For The Treatment Or Prevention Of Flavivirus Infections) for the treatment of or prevention of flavivirus infections	12/19/2002
WO02/100851	The Compounds and methods for (Compounds And Methods For The Treatment Or Prevention Of Flavivirus Infections) for the treatment of or prevention of flavivirus infections	12/19/2002
			5241053	Contain the glycoprotein Gd of HSV-1 and the fusion of LTB (Fused Proteins Comprising Glycoprotein Gd Of HSV-1 And LTB)	8/31/1993
5556946	Interleukin 2/viral antigen albumen chimera (hterleukin-2/Viral Antigen Protein Chimers)	9/17/1996
			6087484	The facilitation oligonucleotides that replaces by 2 '-O-strengthens ribozyme catalysis active (Enhancement Of Ribozyme Catalytic Activity By A 2 '-O-Substituted Facilitator Oligonucleotide)	7/11/2000
5830905	Treat compound, composition and the method (Compounds, Compositions And Methods For Treatment Of Hepatitis C) of the third liver	11/3/1998
			6316492	The method (Methods For Treating Or Preventing Viral Infections And Associated Diseases) for the treatment of or pre-preventing virus infection and relevant disease	11/13/2001
6440985	The method (Methods For Treating Viral Infections) for the treatment of virus infections	8/27/2002
			WO00/10573	Compound, composition and the method (Compounds, Compositions And Methods For Treating Or Preventing Viral Infections And Associated Diseases) for the treatment of or pre-preventing virus infection and relevant disease	3/2/2000
WO00/13708	The method (Methods For Treating Or Preventing Viral Infections And Associated Diseases) for the treatment of or pre-preventing virus infection and relevant disease	3/16/2000
			WO00/18231	The method (Methods For Treating Or Preventing Viral Infections And Associated Diseases) for the treatment of or pre-preventing virus infection and relevant disease	4/6/2000
WO99/51781	Hepatitis C virus Ns5b compoistion and method of use (Hepatitis C Virus Ns5b Compositions And Methods Of Use Thereof)	10/14/1999
			6323180	The third liver inhibitor tripeptides (Hepatitis C Inhibitor Tri-Peptides)	11/27/2001
6143715	The third liver inhibitor peptide analogues (Hepatitis C Inhibitor Peptide Analogues)	11/7/2000
			6329379	The third liver inhibitor tripeptides (Hepatitis C Inhibitor Tri-Peptides)	12/11/2001
6329417	The third liver inhibitor tripeptides (Hepatitis C Inhibitor Tri-Peptides)	12/11/2001
			6410531	The third liver inhibitor tripeptides (Hepatitis C Inhibitor Tri-Peptides)	6/25/2002
6420380	The third liver inhibitor tripeptides (Hepatitis C Inhibitor Tri-Peptides)	7/16/2002
			6448281	Viral polymerase inhibitors (Viral Polymerase Inhibitors)	9/10/2002
6479508	Viral polymerase inhibitors (Viral Polymerase Inhibitors)	11/12/2002
			6534523	The third liver inhibitor tripeptides (Hepatitis C Inhibitor Tri-Peptides)	3/18/2003
WO00/09543	The third liver inhibitor tripeptides (Hepatitis C Inhibitor Tri-Peptides)	2/24/2000
			WO00/09558	The third liver inhibitor peptide (Hepatitis C Inhibitor Peptides)	2/24/2000
WO00/59929	The Macrocyclic peptides of anti-hepatitis C virus active (Macrocyclic Peptides Active Against The Hepatitis C Virus)	10/12/2000
			WO02/04425	Viral polymerase inhibitors (Viral Polymerase Inhibitors)	1/17/2002
WO02/70739	The HCV AG14361 detects (Hcv Polymerase Inhibitor Assay)	9/12/2002
			WO03/07945	Viral polymerase inhibitors (Viral Polymerase Inhibitors)	1/30/2003
WO03/10140	Viral polymerase inhibitors (Viral Polymerase Inhibitors)	2/6/2003
			WO03/10141	Viral polymerase inhibitors (Viral Polymerase Inhibitors)	2/6/2003
WO99/07734	The third liver inhibitor peptide analogues (Hepatitis C Inhibitor Peptide Analogues)	2/18/1999
			WO01/16379	Hepatitis C virus replication inhibitors (Hepatitis C Virus Replication Inhibitors)	3/8/2001
WO02/07761	Suppress the manufacturing of virus-virus and copy (Inhibiting Hepatitis C Virus Processing And Replication)	1/31/2002
			WO02/57287	Nucleoside derivates (Nucleoside Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase) as the RNA viral polymerase inhibitors that depends on RNA	7/25/2002
WO02/57425	Nucleoside derivates (Nucleoside Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase) as the RNA viral polymerase inhibitors that depends on RNA	7/25/2002

WO02/70651	Virus reporter protein particle (Viral Reporter Particles)	9/12/2002
			WO03/20222  PCT/US2003/  041493	Dioxolanes and oxa-mercaptan alkane derivatives are as depending on the RNA viral polymerase inhibitors (Dioxolane And Oxathiolane Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase) of RNA as the thiosemicarbazones (Thiosemicarbazones as Anti-Vitals and Immunopotentiators) of antivirotic and immunopotentiator	3/13/2003   01/10/2003

Table 3: relate to relevant United States Patent (USP) and the disclosed international patent application of suction technology of carrying antiviral compound of the present invention

Publication number	Title	Date of publication
			5740794	The apparatus and method (Apparatus and methods for dispersing dry powder medicaments) of disperseing dry-powder medicament	4/21/1998
5775320	Carry the method and apparatus (Method and device for delivering aerosolized medicaments) of atomization medicine	7/7/1998
			5785049	The method and apparatus (Method and apparatus for dispersion of dry powder medicaments) that disperses dry-powder medicament	7/28/1998
5814607	Lung is carried parathyroid hormone active fragment (Pulmonary delivery of active fragments of parathyroid hormone)	9/29/1998
			5826633	Powder is loaded system, apparatus and method (Powder filling systems, apparatus and methods)	10/27/1998
5458135	Carry the method and apparatus (Method and device for delivering aerosolized medicaments) of atomization medicine	10/17/1995
			5607915	Lung is carried parathyroid hormone active fragment (Pulmonary delivery of active fragments of parathyroid hormone)	3/4/1997
5654007	Processing can disperse the method and system (Methods and system for processing dispersible fine powders) of fine powder	8/5/1997
			5922354	Processing can disperse the method and system (Methods and system for processing dispersible fine powders) of fine powder	7/13/1999
5928469	The method of stored substance (Process for storage of materials)	7/27/1999
			5976574	The method of the organic solvent suspension of spray-drying dewatering medicament (Processes for spray drying hydrophobic drugs in organic solvent suspensions)	11/2/1999
5985248	The method of spray-drying dewatering medicament and composition thereof (Processes for spray drying solutions of hydrophobic drugs and compositions thereof)	11/16/1999
			5994314	Nucleic acid is transported to composition and the method (Compositions and methods for nucleic acid delivery to the lung) of lung	11/30/1999
5997848	Lung is carried the method and composition (Methods and compositions for pulmonary delivery of insulin) of insulin	12/7/1999
			6001336	The method of the aqueous suspensions of spray-drying dewatering medicament and composition thereof (Processes for spray drying aqueous suspensions of hydrophobic drugs and compositions thereof)	12/14/1999
6019968	Dispersibility antibody compositions and preparation and application thereof (Dispersible antibody compositions and methods for their preparation and use)	2/1/2000
			6051256	Dispersibility macromolecule compositions and their preparation and application (Dispersible maeromolecule compositions and methods for their preparation and use)	4/18/2000
6071428	Stable composition (Stable compositions)	6/6/2000
			6077543	Spray-drying contains the system and method (Systems and processes for spray drying hydrophobic drugs with hydrophilic extipients) of the dewatering medicament of hydrophilic excipient	6/20/2000
6080721	Lung is carried parathyroid hormone active fragment (Pulmonary delivery of active fragments of parathyroid hormone)	6/27/2000
			6089228	The apparatus and method (Apparatus and methods for dispersing dry powder medicaments) of disperseing dry-powder medicament	7/18/2000
6103270	Processing can disperse the method and system (Methods and system for processing dispersible fine powders) of fine powder	8/15/2000
			6123936	The method and composition (Methods and compositions for the dry powder formulation of interferons) that is used for the interferon dry-powder preparation	9/26/2000
6136346	Powdered drug preparation (Powdered pharmaceutical formulations having improved dispersibility) with improved dispersiveness	10/24/2000
			6138668	Carry the method and apparatus (Method and device for delivering aerosolized medicaments) of atomization medicine	10/31/2000
6165463	Dispersible antibody compositions and preparation and application thereof (Dispersible antibody compositions and methods for their preparation and use)	12/26/2000
			6182712	Powder filler device and using method thereof (Power filling apparatus and methods for their use)	2/6/2001
6187344	Powdered drug preparation (Powdered pharmaceutical formulations having improved dispersibility) with improved dispersiveness	2/13/2001
			6207135	The gas particles and the manufacture method (Gaseous microparticles for ultrasonic diagnosis and process for their production) thereof that are used for Ultrasonic Diagnosis	3/27/2001
6231851	The method and composition (Methods and compositions for the dry powder formulation of interferons) that is used for the interferon dry-powder preparation	5/15/2001

6257233	Dry powder dispersal device and using method thereof (Dry powder dispersing apparatus and methods for their use)	7/10/2001
			6258341	Stable glassy state powder formulations (Stable glassy state powder formulations)	7/10/2001
6267155	Powder is loaded system, apparatus and method (Powder filling systems, apparatus and methods)	7/31/2001
			6294204	Make the method for the consistent microcapsules of form and the microcapsules (Method of producing morphologically uniform microcapsules and microcapsules produced by this method) of making in this way	9/25/2001
6303582	Carry nucleic acid to composition and the method (Compositions and methods for nucleic acid delivery to the lung) of lung	10/16/2001
			6309623	The stable article (Stabilized preparations for use in metered dose inhalers) that is used for the metering-type inhalator	10/30/2001
6309671	Stable glassy state powder formulations (Stable glassy state powder formulations)	10/30/2001
			6358530	Powdered drug preparation (Powdered pharmaceutical formulations having improved dispersibility) with improved dispersiveness	3/19/2002
6365190	Spray-drying contains the system and method (Systems and processes for spray drying hydrophobic drugs with hydrophilic excipients) of the dewatering medicament of hydrophilic excipient	4/2/2002
			6372258	The method of spray-drying medicine and hydrophobic amino acid (Methods of spray-drying a drug and a hydrophobic amino acid)	4/16/2002
6423344	Dispersible macromolecule compositions and preparation and application thereof (Dispersible macromolecule compositions and methods for their preparation and use)	7/23/2002
			6426210	Material storage (Storage of materials)	7/30/2002
6433040	Stable biologically active goods and using method thereof (Stabilized bioactive preparations and methods of use)	8/13/2002
			6440337	Make the method and apparatus (Method and apparatus for the formation of particles) of particle	8/27/2002
RE37872	Material storage (Storage of materials)	10/8/2002
			6479049	The method and composition (Methods and compositions for the dry powder formulation of interferons) that is used for the interferon dry-powder preparation	11/12/2002
6503411	Stable composition (Stable compositions)	1/7/2003
			6509006	The device of the hydrophobic atomization medicine of lung, composition and method (Devices compositions and methods for the pulmonary delivery of aerosolized medicaments)	1/21/2003
6514496	Dispersible antibody compositions and preparation and application thereof (Dispersible antibody compositions and methods for their preparation and use)	2/4/2003
			6518239	Dry powder composite (dry powder compositions having improved dispersivity) with improved dispersiveness	2/11/2003
6543448	The apparatus and method (apparatus and methods for dispersing dry powder medicaments) of disperseing dry-powder medicament	4/8/2003
			6546929	Dry powder dispersal device and using method thereof (dry powder dispersing apparatus and methods for their use)	4/15/2003
WO 00/15262	The lung of activator of dry powder is carried (dry powder active agent pulmonary delivery)	3/23/2000
			WO 93/00951	Carry the method and apparatus (method and device for delivering aerosolized medicaments) of atomization medicine	1/21/1993
WO 94/07514	Lung is carried parathyroid hormone active fragment (pulmonary delivery of active fragments of parathyroid hormone)	4/14/1994
			WO 95/24183	Lung is carried the method and composition (methods and compositions for pulmonary delivery of insulin) of insulin	9/14/1995
WO 95/31479	The method and composition (methods and compositions for the drypowder formulation of interferons) that is used for the interferon dry-powder preparation	11/23/1995
			WO 96/09085	The apparatus and method (apparatus and methods for dispersing dry powder medicaments) of disperseing dry-powder medicament	3/28/1996
W0 96/32096	Powdered drug preparation (powdered pharmaceutical formulations having improved dispersibility) with improved dispersiveness	10/17/1996
			WO 96/32116	Carry nucleic acid to composition and the method (compositions and methods for nucleic acid delivery to the lung) of lung	10/17/1996
WO 96/32149	The lung of atomization medicine is carried (pulmonary delivery of aerosolized medicaments)	10/17/1996
			WO 96/32152	The lung of dry powder alpha1-antitrypsin is carried (pulmonary administration of dry powder alpha 1-antitrypsin)	10/17/1996
WO 96/40068	Processing can disperse the method and system (methods and system for processing dispersible fine powders) of fine powder	12/19/1996
			WO 97/41031	Powder is loaded system, apparatus and method (powder filling systems, apparatus and methods)	11/6/1997
WO 97/41833	Dispersible macromolecule compositions and preparation and application thereof (dispersible macromolecule compositions and methods for their preparation and use)	11/13/1997
			WO 98/16205	Stable glassy state powder formulations (stable glassy state powder formulations)	4/23/1998
WO 98/29096	The dewatering medicament (aerosolized hydrophobic drug) of atomizing	7/9/1998
			WO 98/29098	Spray-drying contains the method for aqueous suspensions of dewatering medicament of hydrophilic excipient and the composition (processes for spray drying aqueous suspensions of hydrophobic drugs with hydrophilic excipients and compositions prepared by such processes) of making in this way	7/9/1998
WO 98/29140	Spray-drying contains the method and composition (processes and compositions for spray drying hydrophobic drugs in organic solvent suspensions of hydrophilic excipients) of organic solvent suspension of the dewatering medicament of hydrophilic excipient	7/9/1998
			WO 98/29141	Composition (the processes for spray that spray-drying contains the dewatering medicament solution methods of hydrophilic excipient and makes in this way	7/9/1998

	drying solutions of hydrophobic drugs with hydrophilic excipients and compositions prepared by such processes)
			WO 99/19215	Powder filler device and method (powder filling apparatus and method)	4/22/1999
WO 99/42124	The liquid crystal form of cyclosporin (liquid crystal forms of cyclosporin)	8/26/1999
			WO 99/47196	The conveying (aerosolized active agent delivery) of the activating agent of atomizing	9/23/1999
WO 99/62495	Dry powder dispersal device and using method thereof (dry powder dispersing apparatus and methods for their use)	12/9/1999
			WO 00/21594	The conveying (flow resistance modulated aerosolized active agent delivery) of the activating agent of the atomizing that flow resistance is regulated	4/20/2000
WO OO/61178	The dry powder formulations (pulmonary administration of dry powder formulations for treating infertility) that the lung delivering therapeutic is sterile	10/19/2000
			WO 00/72904	The apparatus and method (apparatus and method for dispensing metered amount of aerosolized medication) of metering aerosol dispersion medicine	12/7/2000
WO 01/00263	The system and method for aerosolizing pharmaceutical formulations (systems and methods for aerosolizing pharmaceutical formulations)	1/4/2001
			WO 01/00312	Make the spray drying process (spray drying process for preparing dry powders) of dry powder	1/4/2001
WO 01/32144	Dry powder composite (dry powder compositions having improved dispersivity) with improved dispersiveness	5/10/2001
			WO 01/43529	Be easy to take out the container (receptacles to facilitate the extraction of powders) of powder	6/21/2001
WO 01/43530	Take out from container the system and method (systems and methods for extracting powders from receptacles) of powder	6/21/2001
			WO 01/43802	Process the system and method (systems and methods for treating packaged powders) of packing powder	6/21/2001
WO 01/44764	Non-destructive quality perception system and method (systems and methods for non-destructive mass sensing)	6/21/2001
			WO 01/87393	Open system, the apparatus and method (systems, devices and methods for opening receptacles having a powder to be fluidized) for the treatment of fluidized powder are housed	11/22/2001
WO 01/93932	The locking mechanism of aerosol drug delivery device (lockout mechanism for aerosol drug delivery devices)	12/13/2001
			WO 02/09669	Manufacturing has the apparatus and method of particle of narrow size distribution and the particle (apparatus and process to produce particles having a narrow size distribution and particles made thereby) of making thus	2/7/2002
WO 02/11695	Protein powder (inhaleable spray dried 4-helix bundle protein powders having minimized aggregation) with spray-dired 4-coil wrapping that sucks of minimum cohesion	2/14/2002
			WO 02/49619	Manufacturing contains the induced transformation method (induced phase transition method for the production of microparticles containing hydrophilic active agents) of the particulate of hydrophilic active agent	6/27/2002
WO 02/49620	Manufacturing contains the induced transformation method (induced phase transition method for the production of microparticles containing hydrophobic active agents) of the particulate of hydrophobic active agent	6/27/2002
			WO 02/54868	Lung is carried polyene antifungal agent (pulmonary delivery of polyene antifungal agents)	7/18/2002
WO 02/87542	Carry large molecule to or through new method and the composition (novel methods and compositions for delivering macromolecules to or via the respiratory tract) of respiratory tract	11/7/2002
			WO 02/100548	Process the centrifugal going barrel (centrifuged rotating drum for treating cohesive powders) of cohesive powders	12/19/2002
WO 03/00326	Powder atomization apparatus and method (powder aerosolization apparatus and method)	1/3/2003
			WO 03/00329	The flow regulator of aerosol delivery device and method (flow regulator for aerosol drug delivery device and methods)	1/3/2003

Forward and the reverse primer of table 4:SARSV nucleic acid amplification

Become logarithm	Forward primer SEQ ID NO	The forward primer starting point	The forward primer terminal point	Forward primer Tm	Forward primer %GC	Reverse primer SEQ ID NO	The reverse primer starting point	The reverse primer terminal point	Reverse primer Tm	Reverse primer %GC	Primer Tm Diff	Product length	Product Tm	Product %GC	The annealing scoring	Optimum annealing temperature
																	1	1021	12726	12746	51.3	47.6	3521	12996	12977	50.2	40	1	271	75	42.8	26	52.6
2	1022	12236	12256	51.2	42.9	3522	12993	12975	51.4	47.4	0.2	758	76.4	42.5	26	54
																	3	1023	12373	12391	50.8	47.4	3523	12993	12975	51.4	47.4	0.6	621	76.4	43	26	53.8
4	1024	12236	12256	51.2	42.9	3524	12996	12977	50.2	40	0.9	761	76.4	42.3	26	53.6

Table 5: primer

Forward primer SEQ ID NO and common ordinate		Reverse primer SEQ ID NO and common ordinate		T M(forward and reverse) (℃)		Product length (bp)
							6076	1-19	6171	199-183	50.1	50.3	199
6077	149-169	6172	334-315	51.5	52.4	186
							6078	292-310	6173	560-541	50.8	51.1	269
6079	598-619	6174	749-731	52.6	50.6	152
							6080	721-742	6175	930-912	50.4	50.3	210
6081	888-912	6176	1077-1058	52.8	51.2	190
							6082	984-1003	6177	1149-1131	51.1	51.1	166
6083	1157-1175	6178	1479-1460	50.9	51.6	323
							6084	1420-1441	6179	1700-1680	51.2	50.7	281
6085	1685-1707	6180	1834-1811	53.8	53.7	150
							6086	1740-1764	6181	1987-1963	53.4	52.2	248
6087	2007-2025	6182	2251-2232	50.3	50.1	245
							6088	2226-2245	6183	2385-2366	50.4	50.1	160
6089	2428-2446	6184	2749-2728	50.1	50.3	322
							6090	2742-2763	6185	2893-2875	50.6	51.4	152
6091	2823-2844	6186	3082-3058	50.4	52.3	260
							6092	3007-3031	6187	3185-3164	51.9	51.0	179
6093	3234-3254	6188	3497-3478	51.1	51.3	264
							6094	3453-3476	6189	3647-3627	51.8	52.1	195
6095	3601-3622	6190	3877-3853	52.5	53.6	277
							6096	4007-4027	6191	4158-4135	51.1	51.4	152
6097	4141-4165	6192	4316-4295	51.3	50.8	176
							6098	4366-4387	6193	4567-4544	54.6	55.4	202
6099	4488-4508	6194	4708-4690	50.7	50.3	221
							6100	4658-4677	6195	4994-4974	50.5	51.2	337
6101	4902-4922	6196	5115-5092	50.5	51.4	214
							6102	5239-5260	6197	5450-5430	50.8	50.9	212
6103	5366-5389	6198	5560-5542	50.5	51.8	195
							6104	5593-5612	6199	5860-5836	50.8	51.6	268
6105	6042-6062	6200	6291-6271	50.4	51.1	250
							6106	6271-6291	6201	6483-6463	51.1	50.2	213
6107	7017-7040	6202	7171-7153	52.4	52.8	155
							6108	7253-7272	6203	7504-7486	50.3	50.3	252
6109	7415-7434	6204	7677-7654	54.5	53.6	263
							6110	7615-7635	6205	7821-7798	51.1	52.8	207
6111	7728-7746	6206	7936-7915	51.7	50.1	209
							6112	7845-7867	6207	7994-7970	52.7	53.4	150
6113	8011-8029	6208	8189-8170	51.4	50.6	179
							6114	8143-8166	6209	8300-8281	52.2	50.8	158
6115	8221-8239	6210	8388-8369	51.0	51.1	158
							6116	8553-8575	6211	8931-8915	51.8	50.3	379
6117	8867-8886	6212	9254-9236	50.7	50.6	388
							6118	9244-9267	6213	9597-9573	51.9	53.4	354
6119	9620-9640	6214	9990-9969	51.3	51.3	371
							6120	10009-10027	6215	10188-10171	50.2	50.2	180
6121	10093-10113	6216	10244-10223	52.4	50.6	152
							6122	10242-10265	6217	10608-10589	51.2	51.0	367
6123	10549-10571	6218	10783-10763	53.7	55.2	235

6124	10766-10785	6219	10930-10912	52.0	51.1	165
							6125	11065-11085	6220	11305-11287	50.7	50.0	241
6126	11265-11287	6221	11429-11405	54.5	53.5	165
							6127	11552-11571	6222	11730-11709	52.0	50.4	179
6128	11705-11726	6223	11869-11848	50.1	50.2	165
							6129	11801-11824	6224	11984-11967	51.5	50.4	184
6130	12040-12058	6225	12254-12235	52.3	51.9	215
							6131	12235-12253	6226	12406-12388	50.1	50.1	172
6132	12366-12384	6227	12730-12712	51.7	52.2	365
							6133	12727-12748	6228	12994-12976	50.8	50.3	268
6134	12948-12966	6229	13224-13201	50.7	51.7	277
							6135	13175-13196	6230	13324-13300	54.3	55.1	150
6136	13237-13258	6231	13545-13526	52.9	52.9	309
							6137	13790-13810	6232	13963-13945	50.9	50.7	174
6138	14080-14098	6233	14280-14257	51.5	51.0	201
							6139	14405-14427	6234	14561-14540	50.2	50.9	157
6140	14882-14906	6235	15046-15024	50.9	51.5	165
							6141	14951-14976	6236	15145-15124	53.1	52.9	195
6142	15113-15134	6237	15275-15257	51.6	50.8	163
							6143	15211-15230	6238	15383-15363	50.2	50.1	173
6144	15364-15387	6239	15528-15506	54.0	52.1	165
							6145	15456-15477	6240	15605-15585	52.0	53.2	150
6146	15513-15532	6241	15897-15876	51.2	50.4	385
							6147	15837-15856	6242	15999-15978	52.3	50.8	163
6148	16073-16096	6243	16301-16277	51.7	52.8	229
							6149	16245-16266	6244	16404-16380	50.3	52.0	160
6150	16366-16385	6245	16515-16492	52.9	53.8	150
							6151	16553-16571	6246	16777-16758	53.4	51.5	225
6152	16832-16852	6247	17026-17004	51.0	51.6	195
							6153	16982-17001	6248	17359-17340	51.2	50.2	378
6154	17354-17372	6249	17511-17490	51.3	50.4	158
							6155	17422-17443	6250	17573-17552	50.2	51.1	152
6156	17603-17623	6251	17769-17748	50.7	51.5	167
							6157	17728-17746	6252	17883-17862	50.9	51.2	156
6158	18011-18030	6253	18163-18140	52.9	51.9	153
							6159	18076-18098	6254	18225-18205	54.4	55.0	150
6160	18270-18292	6255	18432-18413	51.9	51.4	163
							6161	18352-18373	6256	18648-18629	51.3	50.8	297
6162	18550-18571	6257	18702-18684	50.4	51.9	153
							6163	18720-18738	6258	19004-18983	50.6	51.0	285
6164	18960-18981	6259	19109-19085	54.7	54.3	150
							6165	19065-19089	6260	19217-19195	52.8	51.7	153
6166	19310-19329	6261	19476-19454	50.2	52.1	167
							6167	19569-19589	6262	19719-19701	50.5	51.8	151
6168	19707-19731	6263	19856-19833	55.7	55.9	150
							6169	19771-19792	6264	19921-19901	50.1	50.2	151
6170	19833-19851	6265	19986-19966	50.9	50.7	154

Table 6: primer

Forward primer SEQ ID NO and common ordinate		Reverse primer SEQ ID NO and common ordinate		T M(forward and reverse) (℃)		Product length (bp)
							6266	20110-20132	6305	20425-20404	51.9	50.9	316

6267	20468-20492	6306	20617-20596	53.2	53.5	150
							6268	20557-20578	6307	20891-20871	50.4	50.6	335
6269	20838-20856	6308	21037-21015	52.5	52.0	200
							6270	21096-21116	6309	21295-21272	50.1	51.7	200
6271	22173-22194	6310	22414-22395	52.4	51.0	242
							6272	22320-22342	6311	22501-22479	54.8	54.3	182
6273	22532-22552	6312	22695-22675	50.6	50.0	164
							6274	22712-22736	6313	22873-22852	56.7	55.5	162
6275	22842-22861	6314	23086-23067	51.0	52.8	245
							6276	23151-23170	6315	23395-23376	51.4	50.3	245
6277	23307-23326	6316	23524-23501	51.1	51.1	218
							6278	23615-23635	6317	23776-23758	50.7	50.2	162
6279	23838-23857	6318	23996-23977	50.4	50.6	159
							6280	24030-24051	6319	24407-24386	57.6	55.7	378
6281	24388-24407	6320	24581-24563	50.4	50.1	194
							6282	24559-24579	6321	24938-24921	52.0	50.4	380
6283	24922-24941	6322	25184-25166	50.1	51.2	263
							6284	25201-25220	6323	25400-25382	51.1	51.4	200
6285	25363-25381	6324	25646-25627	51.1	50.5	284
							6286	25656-25681	6325	25839-25814	54.5	56.4	184
6287	25761-25782	6326	25982-25961	54.6	54.3	222
							6288	26039-26058	6327	26189-26166	54.0	53.0	151
6289	26184-26205	6328	26333-26310	50.9	51.8	150
							6290	26422-26442	6329	26660-26641	51.3	50.2	239
6291	26571-26589	6330	26739-26715	51.7	53.2	169
							6292	26733-26752	6331	26960-26941	51.1	52.2	228
6293	26866-26885	6332	27139-27117	50.7	51.9	274
							6294	27300-27321	6333	27458-27439	51.2	50.2	159
6295	27361-27380	6334	27579-27558	52.4	51.1	219
							6296	27718-27740	6335	27917-27901	50.7	50.0	200
6297	28041-28059	6336	28207-28189	50.8	50.8	167
							6298	28166-28189	6337	28411-28393	52.2	52.9	246
6299	28395-28414	6338	28671-28653	51.5	50.2	277
							6300	28654-28672	6339	28821-28800	50.6	52.3	168
6301	28867-28885	6340	29184-29166	51.5	51.6	318
							6302	29183-29204	6341	29360-29342	50.4	50.4	178
6303	29262-29279	6342	29626-29606	50.1	50.2	365
							6304	29538-29559	6343	29690-29670	50.0	50.4	153

Table 7: primer

Title	SEQ ID NO：	Common ordinate		Title	SEQ ID NO：	Common ordinate
							AB4f	6344	19869-19888		CB1r	6367	28011-28030
AB5f	6345	20238-20257		CB2r	6368	27671-27690
							BC1f	6346	20581-20600		CB3r	6369	27301-27320
BC2f	6347	20950-20969		CB4r	6370	26931-26950
							BC3f	6348	21339-21358		CB5r	6371	26575-26594
BC4f	6349	21708-21727		CB6r	6372	26191-26210
							BC5f	6350	22041-22060		CB7r	6373	25841-25860
BC6f	6351	22410-22429		CB8r	6374	25476-25495
							BC7f	6352	22759-22778		CB9r	6375	25126-25145
BC8f	6353	23131-23150		CB10r	6376	24791-24810
							BC9f	6354	23500-23519		CB11r	6377	24422-24441

BC10f	6355	23841-23860		CB12r	6378	24031-24050
							BC11f	6356	24210-24229		CB13r	6379	23673-23692
BC12f	6357	24560-24579		CB14r	6380	23298-23317
							BC13f	6358	24941-24960		CB15r	6381	22928-22947
BC14f	6359	25310-25329		CB16r	6382	22567-22586
							BC15f	6360	25675-25694		CB17r	6383	22196-22215
BC16f	6361	26044-26063		CB18r	6384	21831-21850
							BC17f	6362	26413-26432		CB19r	6385	21431-21450
BC18f	6363	26763-26782		CB20r	6386	21073-21092
							BC19f	6364	27132-27151		CB21r	6387	20715-20734
BC20f	6365	27491-27510		BA1r	6388	20345-20364
							BC21f	6366	27845-27864		BA2r	6389	19969-19988
				BA3r	6390	19599-19618
											BA4r	6391	19228-19247
				BA5r	6392	18852-18871

Table 8: primer

Title	SEQ ID NO	Common ordinate	Title	SEQ ID NO	Common ordinate
						F1   F2   F3   F4   F5   F6   F7   F8   F9   F10   F11   F12   F13   F14   F15   F16   F17   F18   F19   F20   F21   F22   F23   F24   F25   F26   F27   F28   F29   F30   F31   F32   F33   F34	6393   6394   6395   6396   6397   6398   6399   5400   6401   6402   6403   6404   6405   6406   6407   6408   6409   6410   6411   6412   6413   6414   6415   6416   6417   6418   6419   6420   6421   6422   6423   6424   6425   6426	1-19   292-310   721-742   984-1003   1420-1441   1740-1764   2226-2245   2742-2763   3007-3031   3453-3476   4007-4027   4366-4387   4658-4677   5239-5260   5593-5612   6271-6291   7253-7272   7615-7635   7845-7867   8143-8166   8553-8575   9244-9267   10009-10027   10242-10265   10766-10785   11265-11287   11705-11726   12040-12058   12366-12384   12948-12966   13237-13258   14080-14098   14882-14906   15113-15134	R1   R2   R3   R4   R5   R6   R7   R8   R9   R10   R11   R12   R13   R14   R15   R16   R17   R18   R19   R20   R21   R22   R23   R24   R25   R26   R27   R28   R29   R30   R31   R32   R33   R34	6441   6442   6443   6444   6445   6446   6447   6448   6449   6450   6451   6452   6453   6454   6455   6456   6457   6458   6459   6460   6461   6462   6463   6464   6465   6466   6467   6468   6469   6470   6471   6472   6473   6474	334-315   749-731   1077-1058   1479-1460   1834-1811   2251-2232   2749-2728   3082-3058   3497-3478   3877-3853   4316-4295   4708-4690   5115-5092   5560-5542   6291-6271   7171-7153   7677-7654   7936-7915   8189-8170   8388-8369   9254-9236   9990-9969   10244-10223   10783-10763   11305-11287   11730-11709   11984-11967   12406-12388   12994-12976   13324-13300   13963-13945   14561-14540   15145-15124   15383-15363

F35   F36   F37   F38   F39   F40   F41   F42   F43   F44   F45   F46   F47   F48

6427   6428   6429   6430   6431   6432   6433   6434   6435   6436   6437   6438   6439   6440

15364-15387   15513-15532   16073-16096   16366-16385   16832-16852   17354-17372   17603-17623   18011-18030   18270-18292   18550-18571   18960-18981   19310-19329   19707-19731   19833-19851

R35   R36   R37   R38   R39   R40   R41   R42   R43   R44   R45   R46   R47

6475   6476   6477   6478   6479   6480   6481   6482   6483   6484   6485   6486   6487

15605-15585   15999-15978   16404-16380   16777-16758   17359-17340   17573-17552   17883-17862   18225-18205   18648-18629   19004-18983   19217-19195   19719-19701   19921-19901

Table 9: primer

	Title	SEQ ID NO：
			1	CB12R	6488
2	R0010	6489
			3	R0011	6490
4	R0012	6491
			5	BNI-ED	6492
6	BNI-EU	6493
			7	SAR1S-U	6494
8	SAR1As-D	6495
			9	SAR1S	6496
10	SAR1As	6497
			11	IN2-U	6498
12	IN4-D	6499
			13	IN-2	6500
14	IN-4	6501
			15	IN-6	6502
16	IN-7	6503
			17	COR1-U	6504
18	COR2-D	6505
			19	COR-1	6506
20	COR-2	6507
			21	HKUF-U	6508
22	HKUR-D	6509
			23	HKU-F	6510
24	HKU-R	6511
			25	1451-D	6512
26	1451-U	6513
			27	690-D	6514
28	690-U	6515
			29	690-D2	6516
30	690-U2	6517
			31	EMC7-D	6518
32	EMC7-U	6519
			33	EMC7-D2	6520
34	EMC7-U2	6521
			35	EMC8-D	6522
36	EMC8-U	6523

	Title	SEQ ID NO：
			37	EMC8-D2	6524
38	EMC8-U2	6525
			39	EMC11-D	6526
40	EMC11-U	6527
			41	ORF1B-D	6528
42	ORF1B-U	6529
			43	ORFS-D	6530
44	ORFS-U	6531
			45	E7-717F	6532
46	E8-85R	6533
			47	E8-307F	6534
48	E11-771F	6535
			49	E11-96R	6536
50	CON1-F	6537
			51	CON1-U	6538
52	CON2-F	6539
			53	CON2-R	6540
54	CON3-F	6541
			55	CON3-R	6542
56	15-F	6543
			57	15-R	6544
58	15-F2	6545
			59	15-R2	6546
60	13-F	6547
			61	13-R	6548
62	13-F2	6549
			63	13-R2	6550
64	CONTIG-F	6551
			65	QT3-R	6552
66	QT3-F	6553
			67	QIN-R	6554
68	QIN-F	6555
			69	AB1-F	6556
70	AB2-F	6557
			71	AB3-F	6558
72	AB1-R	6559

The feature of table 10:SARS virus predicted protein matter and ORF

	SARS ORF   (SEQ ID NO)	Length (aa)	Effect	Cleavage site	Feature	Cons d*
							ORF1a	P28(9766)	179	Leader albumen	179(G/G) #		+
P65(9767)	639	MHV p65 cleaved products homologue	818(G/A)		+
						Nsp1   (9768)		2422 ##	Papain sample protease, cutting the first two protein	3240(Q/S)	The sulfatase domain, zinc-binding domain	+
Nsp2   (9769)	306	3C sample protease, scinderin nsp1-nsp12	3546(Q/G)		+
						Nsp3(9770)		290	？	3836(Q/S)	5 TMDs	+
Nsp4(9771)	83	？	3919(Q/A)	1 TMD	+
						Nsp5(9772)		198	？	4117(Q/N)		+
Nsp6(9773)	113	？	4230(Q/A)		+
						Nsp7(9774)		139	？	4369(Q/S)	Imaginary growth factor-like motif	+
ORF1b	Nsp9(9775)	932	RNA polymerase	5298(Q/A)									+
						Nsp10(9776)	601	The helicase Tanner that supposes etc., (2003) J Biol Chem 278:39578-82	5899(Q/A)	Metal binding domain, ATP/GTP is in conjunction with the territory	+
	Nsp11(9777)	527	？	6426(Q/S)								+
						Nsp12(9778)	346	？	6772(Q/A)		+
	Nsp13(9779)	298	？	-								+
						Structural area	Furcella (S) (6042)		The major antigen determinant contains receptor binding domains		The leader peptide, 1 TMD, 17 N-glycosylation sites		+
Orf3(6043)	274	？		2 TMD, 1 N-glycosylation site, 10 O-glycosylation sites	-
							Orf4(6044)	154	？			-
Coating (E) (6045)	76	Relevant with peplos		1 TMD, 2 N-glycosylation sites	+
							Matrix (M) (6046)	221	Relevant with peplos, transmembrane protein		3 TMD, 1 N-glycosylation site	+
Orf7   (6047)	63	？		1 TMD	-
							Orf8(6048)	122	？		1 TMD	-
Orf9(6049)	44	？		Surface-associated	-
							Orf10	39	？		Surface-associated	-
Orf11(6050)	84	？		1 N-glycosylation site	-
							Nucleocapsid (N) (6052)	422	Related with virus genome RNA		Phosphoprotein	+
Orf13	98	？		1 O-glycosylation site	-

TMD: the membrane spaning domain of prediction.

Cons ^d* :+be illustrated at least a other coronavirus and have corresponding albumen

#: perhaps (namely at G/A) cutting obtains a 180mer product after Gly-Gly

##: this 2422mer product also can be cut afterwards at residue 1922 (Gly-2740 of SEQ ID NO:6039) and obtain one and comprise the Zn binding motif

The 1922mer product P lpro of (SEQ ID NO:7254) and a 500mer product.

Protein homology between table 11:SARS and other coronavirus

The ratio of amino acid homogeny between numeral SARS albumen and other coronavirus corresponding gene product. More conservative to representing with black matrix;

More variable to representing with underscore.

	Group 1			Group 2		Group 3
							Protein	229E	TGV	PEDV	MHV	BCoV	AIBV
The replicase zone
							Leader albumen p28 p65 homologue nsp1 (PLP protease) nsp2 (3CL protease) nsp3 nsp4 nsp5 nsp6 nsp7	＜20   ＜20   25.5   40.4   30   38.6   48.2   45.1   53.8	＜20   23   25.8   43.8   27   42.2   42.9   38.9   54.5	＜20   23   25.4   44.6   29.4   39.8   43.9   45.1   56.1	27   ＜20   29   50   34.2   47.5   46.8   45.1   56.2	＜20   20   30   48.4   35.5   46.1   47.3   46.9   55.4	＜20   ＜20   25   41   28.5   37.3   38.7   39.8   58.3
Nsp9 (polymerase) nsp10 (helicase) nsp11 nsp12 nsp13	59.8   60.7   52.3   43.1   56.4	59.6   62   53.7   43   54.4	60   62.3   52.3   45.4   55.3	67.3   67.2   57.6   45.9   63	66.9   68.6   57.6   45   65	62.4   58.9   52   40.2   53.4
							Structural area
Furcella (S) coating (E) matrix glycoprotein (M) nucleocapsid (N)	28.8   33*   30.6   26.9	31.6*   27.9   32.5   30.1	30.3   20   34.8   29.5	31.1   23   40.8   37.3	31   26.5   41.9   37.4	32.7*   23.2   32.5   31.5

* these three contrasts only obtain in a fragment of holoprotein.

Table 12: five kinds of SARS separator nucleotides and amino acid whose difference

		FRA*	TOR2*	Urbani*	CUHK*	HKU*
								Position °	Base/amino acid	Base/amino acid	Base/amino acid	Base/amino acid	Base/amino acid
							ORF1a	2557	A/Thr	G/Ala	G/Ala	G/Ala	G/Ala
2601	T/Val				C
						7746		G/Pro			T
7919	C/Ala		T/Val
						7930		G/Asp				A/Asn
8387	G/Ser				C/Thr
						8416		G/Arg				C/Thr
9404	T/Val			C/Ala
						9479		T/Val			C/Ala
11448	T/Ile	C	C	C	C
ORF1b	13494	GT/Val					AG/Ser
						16622		C/Ala		T
	17564	T/Asp			C/Glu
						17846		C/Arg			T
	18065	G/Lys					A
						18965		A/Ile	T	T	T	T
	19064	A/Glu		G	G
						19084		T/Ile	C/Thr	C/Thr	C/Thr	C/Thr
Furcella						21721		G/Gly			A/Asp
	22222	T/Ile			C/Thr
						23220		T/Ser	G/Ala
	24872	T/Leu		C
						24933		T/Phe	C/Leu	C/Leu	C/Leu	C/Leu
ORF3						25298		G/Gly	A/Arg
	25569	T/Met					A/Lys
Matrix	26600	T/Val	C/Ala	C/Ala	C/Ala
						26857		T/Ser		C/Pro
ORF10						27827		T/Cys			C/Arg
Nucleocapsid						28268		T/Ile	C/Thr	C/Thr	C/Thr	C/Thr

* sars coronavirus FRA (accession number AY310120)

* sars coronavirus TOR2 (accession number AY274119)

* sars coronavirus Urbani (accession number AY278741)

* sars coronavirus CUHK-W1 (accession number AY278554)

* sars coronavirus HKU-39849 (accession number AY278491)

° this position is based on the FRA sequence.

Show the T Antigen Epitope Prediction of 13-25:SEQ ID NOS:6039-6050 and 6052

Antigen Epitope Prediction carries out at http://www.mpiib-berlin.mpg.de/MAPPP/binding.html, uses minimum to score 0.5 and BIMAS matrix, selects maximum among 20 results one. This analysis has disclosed the epi-position of 9mer and 10mer.

The epi-position of table 13:SEQ ID NO:6039

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1867	SEQ ID NO：7400	8％	450
2	4139	SEQ ID NO：7401	5.55％	312.5
					3	88	SEQ ID NO：7402	4％	225
4	4249	SEQ ID NO：7403	3.55％	200
					5	4059	SEQ ID NO：7404	2.22％	125
6	2027	SEQ ID NO：7405	1.6％	90
					7	3413	SEQ ID NO：7406	1.11％	62.5
8	1823	SEQ ID NO：7407	0.88％	50
					9	2798	SEQ ID NO：7408	0.88％	50
10	220	SEQ ID NO：7409	0.8％	45
					11	3738	SEQ ID NO：7410	0.8％	45
12	4182	SEQ ID NO：7411	0.8％	45
					13	4174	SEQ ID NO：7412	0.66％	37.5
14	1940	SEQ ID NO：7413	0.55％	31.25
					15	38	SEQ ID NO：7414	0.48％	27
16	1231	SEQ ID NO：7415	0.44％	25
					17	1613	SEQ ID NO：7416	0.44％	25
18	3645	SEQ ID NO：7417	0.44％	25
					19	4192	SEQ ID NO：7418	0.44％	25
20	378	SEQ ID NO：7419	0.4％	22.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1867	SEQ ID NO：7420	8％	450
2	1495	SEQ ID NO：7421	4％	225
					3	3921	SEQ ID NO：7422	2.4％	135
4	486	SEQ ID NO：7423	2.22％	125
					5	4139	SEQ ID NO：7424	2.22％	125
6	62	SEQ ID NO：7425	1.6％	90
					7	1190	SEQ ID NO：7426	1.6％	90
8	1284	SEQ ID NO：7427	1.6％	90
					9	3284	SEQ ID NO：7428	1.6％	90
10	2921	SEQ ID NO：7429	1.2％	67.5

11	349	SEQ ID NO：7430	0.8％	45
					12	789	SEQ ID NO：7431	0.8％	45
13	1185	SEQ ID NO：7432	0.8％	45
					14	4184	SEQ ID NO：7433	0.8％	45
15	1313	SEQ ID NO：7434	0.64％	36
					16	3948	SEQ ID NO：7435	0.48％	27
17	149	SEQ ID NO：7436	0.44％	25
					18	941	SEQ ID NO：7437	0.44％	25
19	1390	SEQ ID NO：7438	0.44％	25
					20	1613	SEQ ID NO：7439	0.44％	25

HLA A3-9 mers
					Maximum possible with this molecule type is scored				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1010	SEQ ID NO：7440	1.48％	180
2	3155	SEQ ID NO：7441	1.48％	180
					3	1229	SEQ ID NO：7442	1.23％	150
4	2405	SEQ ID NO：7443	0.88％	108
					5	2	SEQ ID NO：7444	0.74％	90
6	2304	SEQ ID NO：7445	0.74％	90
					7	2358	SEQ ID NO：7446	0.74％	90
8	3160	SEQ ID NO：7447	0.74％	90
					9	3771	SEQ ID NO：7448	0.74％	90
10	4007	SEQ ID NO：7449	0.74％	90
					11	3079	SEQ ID NO：7450	0.66％	81
12	4045	SEQ ID NO：7451	0.66％	81
					13	1081	SEQ ID NO：7452	0.49％	60
14	3268	SEQ ID NO：7453	0.49％	60
					15	4144	SEQ ID NO：7454	0.49％	60
16	614	SEQ ID NO：7455	0.37％	45
					17	728	SEQ ID NO：7456	0.37％	45
18	1537	SEQ ID NO：7457	0.37％	45
					19	313	SEQ ID NO：7458	0.32％	40
20	1744	SEQ ID NO：7459	0.32％	40

HLA A3-10 mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	62	SEQ ID NO：7460	4.44％	540
2	2151	SEQ ID NO：7461	2.46％	300
					3	633	SEQ ID NO：7462	2.22％	270
4	1158	SEQ ID NO：7463	2.22％	270

5	2565	SEQ ID NO：7464	2.22％	270
					6	2298	SEQ ID NO：7465	1.77％	216
7	3159	SEQ ID NO：7466	1.11％	135
					8	640	SEQ ID NO：7467	0.98％	120
9	2186	SEQ ID NO：7468	0.74％	90
					10	3869	SEQ ID NO：7469	0.74％	90
11	2308	SEQ ID NO：7470	0.66％	81
					12	786	SEQ ID NO：7471	0.55％	67.5
13	749	SEQ ID NO：7472	0.49％	60
					14	1080	SEQ ID NO：7473	0.49％	60
15	2358	SEQ ID NO：7474	0.49％	60
					16	3955	SEQ ID NO：7475	0.49％	60
17	714	SEQ ID NO：7476	0.37％	45
					18	1081	SEQ ID NO：7477	0.37％	45
19	1170	SEQ ID NO：7478	0.37％	45
					20	1228	SEQ ID N0：7479	0.37％	45

HLA A24-9 mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	3797	SEQ ID NO：7480	37.57％	600
2	4202	SEQ ID NO：7481	37.57％	600
					3	3189	SEQ ID NO：7482	25.05％	400
4	1864	SEQ ID NO：7483	23.14％	369.6
					5	1066	SEQ ID NO：7484	22.54％	360
6	2143	SEQ ID NO：7485	22.54％	360
					7	2693	SEQ ID NO：7486	22.54％	360
8	1426	SEQ ID NO：7487	18.78％	300
					9	1238	SEQ ID NO：7488	18.03％	288
10	3768	SEQ ID NO：7489	18.03％	288
					11	797	SEQ ID NO：7490	15.03％	240
12	1882	SEQ ID NO：7491	15.03％	240
					13	1490	SEQ ID NO：7492	13.77％	220
14	2237	SEQ ID NO：7493	13.77％	220
					15	95	SEQ ID NO：7494	12.52％	200
16	1821	SEQ ID NO：7495	12.52％	200
					17	2289	SEQ ID NO：7496	12.52％	200
18	3080	SEQ ID NO：7497	12.52％	200
					19	3660	SEQ ID NO：7498	12.52％	200
20	4354	SEQ ID NO：7499	12.52％	200

HLA A24-10 mers

Use the maximum possible of this molecule type to score				1596.672
					Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
1	2143	SEQ ID NO：7500	37.87％	604.8
					2	1159	SEQ ID NO：7501	26.30％	420
3	1650	SEQ ID NO：7502	26.30％	420
					4	1150	SEQ ID NO：7503	18.78％	300
5	2763	SEQ ID NO：7504	18.78％	300
					6	3165	SEQ ID NO：7505	18.78％	300
7	3201	SEQ ID NO：7506	15.03％	240
					8	3694	SEQ ID NO：7507	15.03％	240
9	4204	SEQ ID NO：7508	15.03％	240
					10	1692	SEQ ID NO：7509	13.77％	220
11	797	SEQ ID NO：7510	12.52％	200
					12	1610	SEQ ID NO：7511	12.52％	200
13	1789	SEQ ID NO：7512	12.52％	200
					14	1881	SEQ ID NO：7513	12.52％	200
15	3090	SEQ ID NO：7514	12.52％	200
					16	3763	SEQ ID NO：7515	12.52％	200
17	2569	SEQ ID NO：7516	11.27％	180
					18	194	SEQ ID NO：7517	9.39％	150
19	1771	SEQ ID NO：7518	9.39％	150
					20	2488	SEQ ID NO：7519	9.39％	150

HLA A 0201-9 mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	2308	SEQ ID NO：7520	0.20％	8144.13515256
2	3729	SEQ ID NO：7521	0.10％	4047.23088
					3	3574	SEQ ID NO：7522	0.09％	3547.4996634
4	3615	SEQ ID NO：7523	0.06％	2722.682592
					5	3159	SEQ ID NO：7524	0.05％	1999.734264
6	2339	SEQ ID NO：7525	0.03％	1551.92907744
					7	2201	SEQ ID NO：7526	0.03％	1521.53694
8	3559	SEQ ID NO：7527	0.02％	1174.38939504
					9	3085	SEQ ID NO：7528	0.02％	1146.296448
10	4070	SEQ ID NO：7529	0.02％	970.4103696
					11	3708	SEQ ID NO：7530	0.02％	958.92888
12	3098	SEQ ID NO：7531	0.02％	942.678
					13	1362	SEQ ID NO：7532	0.02％	900.6984
14	3563	SEQ ID NO：7533	0.01％	735.86016
					15	3774	SEQ ID NO：7534	0.01％	687.655656
16	4242	SEQ ID NO：7535	0.01％	685.78272

17	2340	SEQ ID NO：7536	0.01％	668.37342936
					18	650	SEQ ID NO：7537	0.01％	640.1983392
19	3862	SEQ ID NO：7538	0.01％	620.57772
					20	2860	SEQ ID NO：7539	0.01％	607.88448

HLA A 0201-10 mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	2307	SEQ ID NO：7540	0.40％	15915.66281448
2	2201	SEQ ID NO：7541	0.12％	4772.09313
					3	3558	SEQ ID NO：7542	0.05％	2295.04855632
4	1772	SEQ ID NO：7543	0.04％	1759.6656
					5	3087	SEQ ID NO：7544	0.03％	1215.76896
6	2339	SEQ ID NO：7545	0.02％	1116.29986272
					7	2308	SEQ ID NO：7546	0.02％	970.14776112
8	3061	SEQ ID NO：7547	0.02％	836.2525104
					9	2748	SEQ ID NO：7548	0.01％	726.706344
10	3837	SEQ ID NO：7549	0.01％	720.8292
					11	59	SEQ ID NO：7550	0.01％	650.3112
12	2877	SEQ ID NO：7551	0.01％	620.22996
					13	4114	SEQ ID NO：7552	0.01％	559.8936
14	805	SEQ ID NO：7553	0.01％	484.4565072
					15	1655	SEQ ID NO：7554	0.01％	437.48208
16	611	SEQ ID NO：7555	0.00％	319.9392
					17	1961	SEQ ID NO：7556	0.00％	305.94186
18	1223	SEQ ID NO：7557	0.00％	289.08792
					19	852	SEQ ID NO：7558	0.00％	285.67242
20	2139	SEQ ID NO：7559	0.00％	284.845869

HLA A 1101-9 mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	4200	SEQ ID NO：7560	50％	18
2	281	SEQ ID NO：7561	25％	9
					3	3236	SEQ ID NO：7562	25％	9
4	509	SEQ ID NO：7563	16.66％	6
					5	848	SEQ ID NO：7564	16.66％	6
6	2193	SEQ ID NO：7565	16.66％	6
					7	3542	SEQ ID NO：7566	16.66％	6
8	541	SEQ ID NO：7567	15％	5.4
					9	1748	SEQ ID NO：7568	12.5％	4.5
10	829	SEQ ID NO：7569	11.11％	4

11	1149	SEQ ID NO：7570	11.11％	4
					12	2027	SEQ ID NO：7571	11.11％	4
13	2576	SEQ ID NO：7572	11.11％	4
					14	873	SEQ ID NO：7573	8.33％	3
15	2725	SEQ ID NO：7574	8.33％	3
					16	3541	SEQ ID NO：7575	8.33％	3
17	1837	SEQ ID NO：7576	7.5％	2.7
					18	2475	SEQ ID NO：7577	7.5％	2.7
19	2703	SEQ ID NO：7578	7.5％	2.7
					20	1823	SEQ ID NO：7579	6.66％	2.4

HLA A 1101-10 mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	3541	SEQ ID NO：7580	50％	18
2	281	SEQ ID NO：7581	25％	9
					3	1495	SEQ ID NO：7582	25％	9
4	2303	SEQ ID NO：7583	25％	9
					5	2616	SEQ ID NO：7584	25％	9
6	48	SEQ ID NO：7585	16.66％	6
					7	1394	SEQ ID NO：7586	16.66％	6
8	1499	SEQ ID NO：7587	16.66％	6
					9	1862	SEQ ID NO：7588	16.66％	6
10	1163	SEQ ID NO：7589	11.11％	4
					11	4006	SEQ ID NO：7590	11.11％	4
12	4344	SEQ ID NO：7591	11.11％	4
					13	633	SEQ ID NO：7592	10％	3.6
14	119	SEQ ID NO：7593	8.33％	3
					15	1190	SEQ ID NO：7594	8.33％	3
16	1195	SEQ ID NO：7595	8.33％	3
					17	1725	SEQ ID NO：7596	8.33％	3
18	2728	SEQ ID NO：7597	8.33％	3
					19	2895	SEQ ID NO：7598	8.33％	3
20	3033	SEQ ID NO：7599	8.33％	3

HLA B7-9 mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1335	SEQ ID NO：7600	4.44％	240
2	2580	SEQ ID NO：7601	4.44％	240
					3	1703	SEQ ID NO：7602	3.70％	200
4	113	SEQ ID NO：7603	2.22％	120

5	168	SEQ ID NO：7604	2.22％	120
					6	2842	SEQ ID NO：7605	2.22％	120
7	4027	SEQ ID NO：7606	2.22％	120
					8	3680	SEQ ID NO：7607	1.66％	90
9	2085	SEQ ID NO：7608	1.48％	80
					10	2492	SEQ ID NO：7609	1.48％	80
11	2660	SEQ ID NO：7610	1.48％	80
					12	2906	SEQ ID NO：7611	1.48％	80
13	3346	SEQ ID NO：7612	1.48％	80
					14	4038	SEQ ID NO：7613	1.48％	80
15	1163	SEQ ID NO：7614	1.11％	60
					16	1457	SEQ ID NO：7615	1.11％	60
17	2351	SEQ ID NO：7616	1.11％	60
					18	2471	SEQ ID NO：7617	1.11％	60
19	3499	SEQ ID NO：7618	1.11％	60
					20	3635	SEQ ID NO：7619	1.11％	60

HLA B7-10 mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1703	SEQ ID NO：7620	3.70％	200
2	17	SEQ ID NO：7621	2.22％	120
					3	3008	SEQ ID NO：7622	2.22％	120
4	4106	SEQ ID NO：7623	2.22％	120
					5	3450	SEQ ID NO：7624	1.66％	90
6	113	SEQ ID NO：7625	1.48％	80
					7	195	SEQ ID NO：7626	1.48％	80
8	307	SEQ ID NO：7627	1.48％	80
					9	780	SEQ ID NO：7628	1.48％	80
10	1000	SEQ ID NO：7629	1.48％	80
					11	1072	SEQ ID NO：7630	1.48％	80
12	1404	SEQ ID NO：7631	1.48％	80
					13	1980	SEQ ID NO：7632	1.48％	80
14	2262	SEQ ID NO：7633	1.48％	80
					15	2543	SEQ ID NO：7634	1.48％	80
16	2906	SEQ ID NO：7635	1.48％	80
					17	3077	SEQ ID NO：7636	1.48％	80
18	3175	SEQ ID NO：7637	1.48％	80
					19	4195	SEQ ID NO：7638	1.48％	80
20	4251	SEQ ID NO：7639	1.48％	80

The epi-position of table 14:SEQ ID NO:6040

HLA A1-9 mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	20	SEQ ID NO：7640	0.04％	2.25
2	91	SEQ ID NO：7641	0.01％	1
					3	125	SEQ ID NO：7642	0.01％	0.75
4	56	SEQ ID NO：7643	0.00％	0.5
					5	145	SEQ ID NO：7644	0.00％	0.5

HLA A1-10 mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	20	SEQ ID NO：7645	0.01％	0.9
2	56	SEQ ID NO：7646	0.00％	0.5
					3	71	SEQ ID NO：7647	0.00％	0.5
4	144	SEQ ID NO：7648	0.00％	0.5

HLA A3-9 mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	115	SEQ ID NO：7649	0.24％	30
2	87	SEQ ID NO：7650	0.04％	6
					3	80	SEQ ID NO：7651	0.03％	4.05
4	125	SEQ ID NO：7652	0.01％	1.8
					5	39	SEQ ID NO：7653	0.01％	1.5
6	56	SEQ ID NO：7654	0.01％	1.5
					7	135	SEQ ID NO：7655	0.00％	1.2
8	91	SEQ ID NO：7656	0.00％	1
					9	119	SEQ ID NO：7657	0.00％	1
10	141	SEQ ID NO：7658	0.00％	0.9
					11	150	SEQ ID NO：7659	0.00％	0.6
12	137	SEQ ID NO：7660	0.00％	0.54

HLA A3-10 mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	36	SEQ ID NO：7661	0.24％	30
2	144	SEQ ID NO：7662	0.06％	8
					3	101	SEQ ID NO：7663	0.03％	4
4	99	SEQ ID NO：7664	0.02％	3.6
					5	80	SEQ ID NO：7665	0.02％	2.7
6	125	SEQ ID NO：7666	0.01％	1.6875

7	71	SEQ ID NO：7667	0.01％	1.5
					8	118	SEQ ID NO：7668	0.01％	1.5
9	40	SEQ ID NO：7669	0.01％	1.35
					10	5	SEQ ID NO：7670	0.00％	0.9
11	56	SEQ ID NO：7671	0.00％	0.9
					12	107	SEQ ID NO：7672	0.00％	0.6
13	135	SEQ ID NO：7673	0.00％	0.6
					14	141	SEQ ID NO：7674	0.00％	0.6
15	148	SEQ ID NO：7675	0.00％	0.6
					16	116	SEQ ID NO：7676	0.00％	0.5

HLA A24-9 mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	153	SEQ ID NO：7677	1.05％	16.8
2	80	SEQ ID NO：7678	0.75％	12
					3	123	SEQ ID NO：7679	0.50％	8
4	137	SEQ ID NO：7680	0.50％	8
					5	9	SEQ ID NO：7681	0.45％	7.2
6	77	SEQ ID NO：7682	0.45％	7.2
					7	112	SEQ ID NO：7683	0.45％	7.2
8	73	SEQ ID NO：7684	0.41％	6.6
					9	32	SEQ ID NO：7685	0.37％	6
10	110	SEQ ID NO：7686	0.37％	6
					11	140	SEQ ID NO：7687	0.37％	6
12	143	SEQ ID NO：7688	0.37％	6
					13	18	SEQ ID NO：7689	0.30％	4.8
14	54	SEQ ID NO：7690	0.30％	4.8
					15	108	SEQ ID NO：7691	0.30％	4.8
16	141	SEQ ID NO：7692	0.30％	4.8
					17	92	SEQ ID NO：7693	0.27％	4.4
18	33	SEQ ID NO：7694	0.25％	4
					19	49	SEQ ID NO：7695	0.25％	4
20	111	SEQ ID NO：7696	0.25％	4

HLA A24-10 mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	142	SEQ ID NO：7697	12.52％	200
2	110	SEQ ID NO：7698	0.75％	12
					3	99	SEQ ID NO：7699	0.50％	8
4	8	SEQ ID NO：7700	0.45％	7.2

5	140	SEQ ID NO：7701	0.45％	7.2
					6	32	SEQ ID NO：7702	0.37％	6
7	17	SEQ ID NO：7703	0.30％	4.8
					8	53	SEQ ID NO：7704	0.30％	4.8
9	76	SEQ ID NO：7705	0.30％	4.8
					10	107	SEQ ID NO：7706	0.30％	4.8
11	111	SEQ ID NO：7707	0.30％	4.8
					12	72	SEQ ID NO：7708	0.27％	4.4
13	91	SEQ ID NO：7709	0.27％	4.4
					14	31	SEQ ID NO：7710	0.25％	4
15	127	SEQ ID NO：7711	0.25％	4
					16	139	SEQ ID NO：7712	0.25％	4
17	80	SEQ ID NO：7713	0.22％	3.6
					18	38	SEQ ID NO：7714	0.18％	3
19	118	SEQ ID NO：7715	0.18％	3
					20	49	SEQ ID NO：7716	0.12％	2

HLA A 0201-9 mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	80	SEQ ID NO：7717	0.00％	171.96732
2	147	SEQ ID NO：7718	0.00％	51.46848
					3	143	SEQ ID NO：7719	0.00％	11.6146182
4	56	SEQ ID NO：7720	0.00％	11.304684
					5	10	SEQ ID NO：7721	0.00％	10.34586
6	6	SEQ ID NO：7722	0.00％	6.56830734
					7	26	SEQ ID NO：7723	0.00％	6.07614
8	141	SEQ ID NO：7724	0.00％	5.981472
					9	148	SEQ ID NO：7725	0.00％	5.194044
10	9	SEQ ID NO：7726	0.00％	4.299183
					11	137	SEQ ID NO：7727	0.00％	4.299183
12	130	SEQ ID NO：7728	0.00％	4.138344
					13	84	SEQ ID NO：7729	0.00％	3.42792
14	27	SEQ ID NO：7730	0.00％	3.383484
					15	2	SEQ ID NO：7731	0.00％	3.381
16	62	SEQ ID NO：7732	0.00％	3.251556
					17	23	SEQ ID NO：7733	0.00％	2.9542005
18	99	SEQ ID NO：7734	0.00％	1982232
					19	33	SEQ ID NO：7735	0.00％	1.86921
20	111	SEQ ID NO：7736	0.00％	1.76402985

HLA A 0201-10 mers

Use the maximum possible of this molecule type to score				3925227.1
					Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
1	5	SEQ ID NO：7737	0.00％	159.9696
					2	25	SEQ ID NO：7738	0.00％	69.552
3	80	SEQ ID NO：7739	0.00％	36.5148
					4	107	SEQ ID NO：7740	0.00％	21.3624
5	148	SEQ ID NO：7741	0.00％	17.73576
					6	61	SEQ ID NO：7742	0.00％	13.9104
7	147	SEQ ID NO：7743	0.00％	11.304684
					8	53	SEQ ID NO：7744	0.00％	8.230458
9	17	SEQ ID NO：7745	0.00％	7.3086111
					10	110	SEQ ID NO：7746	0.00％	6.174104475
11	9	SEQ ID NO：7747	0.00％	6.0858
					12	99	SEQ ID NO：7748	0.00％	5.6823984
13	2	SEQ ID NO：7749	0.00％	3.188283
					14	41	SEQ ID NO：7750	0.00％	2.206413
15	135	SEQ ID NO：7751	0.00％	2.076624
					16	76	SEQ ID NO：7752	0.00％	2.005692
17	23	SEQ ID NO：7753	0.00％	1.798209
					18	40	SEQ ID NO：7754	0.00％	1.68996456
19	39	SEQ ID NO：7755	0.00％	1.516482
					20	118	SEQ ID NO：7756	0.00％	1.2683304

HLA A 1101-9 mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	91	SEQ ID NO：7757	2.77％	1

HLA A 1101-10 mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	101	SEQ ID NO：7758	33.33％	12
2	71	SEQ ID NO：7759	2.77％	1
					3	90	SEQ ID NO：7760	1.66％	0.6

HLA B7-9 mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	49	SEQ ID NO：7761	2.22％	120
2	9	SEQ ID NO：7762	1.11％	60
					3	73	SEQ ID NO：7763	0.66％	36
4	33	SEQ ID NO：7764	0.37％	20

5	137	SEQ ID NO：7765	0.37％	20
					6	141	SEQ ID NO：7766	0.37％	20
7	77	SEQ ID NO：7767	0.22％	12
					8	112	SEQ ID NO：7768	0.22％	12
9	143	SEQ ID NO：7769	0.22％	12
					10	81	SEQ ID NO：7770	0.14％	8
11	13	SEQ ID NO：7771	0.09％	5
					12	69	SEQ ID NO：7772	0.09％	5
13	18	SEQ ID NO：7773	0.07％	4
					14	32	SEQ ID NO：7774	0.07％	4
15	54	SEQ ID NO：7775	0.07％	4
					16	80	SEQ ID NO：7776	0.07％	4
17	92	SEQ ID NO：7777	0.07％	4
					18	108	SEQ ID NO：7778	0.07％	4
19	111	SEQ ID NO：7779	0.07％	4
					20	123	SEQ ID NO：7780	0.07％	4

HLA B7-10 mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	99	SEQ ID NO：7781	0.74％	40
2	17	SEQ ID NO：7782	0.37％	20
					3	8	SEQ ID NO：7783	0.22％	12
4	72	SEQ ID NO：7784	0.22％	12
					5	91	SEQ ID NO：7785	0.22％	12
6	127	SEQ ID NO：7786	0.11％	6
					7	31	SEQ ID NO：7787	0.07％	4
8	32	SEQ ID NO：7788	0.07％	4
					9	53	SEQ ID NO：7789	0.07％	4
10	76	SEQ ID NO：7790	0.07％	4
					11	107	SEQ ID NO：7791	0.07％	4
12	110	SEQ ID NO：7792	0.07％	4
					13	111	SEQ ID NO：7793	0.07％	4
14	140	SEQ ID NO：7794	0.07％	4
					15	9	SEQ ID NO：7795	0.05％	3
16	19	SEQ ID NO：7796	0.05％	3
					17	33	SEQ ID NO：7797	0.03％	2
18	93	SEQ ID NO：7798	0.03％	2
					19	102	SEQ ID NO：7799	0.03％	2
20	129	SEQ ID NO：7800	0.02％	1.5

The epi-position of table 15:SEQ ID NO:6041

HLA A1-9 mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1818	SEQ ID NO：7801	1.6％	90
2	373	SEQ ID NO：7802	1.33％	75
					3	681	SEQ ID NO：7803	1.33％	75
4	74	SEQ ID NO：7804	0.88％	50
					5	786	SEQ ID NO：7805	0.88％	50
6	1495	SEQ ID NO：7806	0.88％	50
					7	88	SEQ ID NO：7807	0.8％	45
8	357	SEQ ID NO：7808	0.8％	45
					9	1271	SEQ ID NO：7809	0.8％	45
10	1799	SEQ ID NO：7810	0.8％	45
					11	1393	SEQ ID NO：7811	0.48％	27
12	386	SEQ ID NO：7812	0.44％	25
					13	2304	SEQ ID NO：7813	0.44％	25
14	198	SEQ ID NO：7814	0.4％	22.5
					15	840	SEQ ID NO：7815	0.4％	22.5
16	2359	SEQ ID NO：7816	0.4％	22.5
					17	1194	SEQ ID NO：7817	0.32％	18
18	1546	SEQ ID NO：7818	0.32％	18
					19	2200	SEQ ID NO：7819	0.22％	12.5
20	996	SEQ ID NO：7820	0.2％	11.25

HLA A1-10 mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	995	SEQ ID NO：7821	10％	562.5
2	1303	SEQ ID NO：7822	2.22％	125
					3	1582	SEQ ID NO：7823	2％	112.5
4	1456	SEQ ID NO：7824	1.6％	90
					5	772	SEQ ID NO：7825	1.11％	62.5
6	181	SEQ ID NO：7826	0.88％	50
					7	632	SEQ ID NO：7827	0.88％	50
8	2281	SEQ ID NO：7828	0.88％	50
					9	1586	SEQ ID NO：7829	0.8％	45
10	2109	SEQ ID NO：7830	0.8％	45
					11	745	SEQ ID NO：7831	0.55％	31.25
12	1916	SEQ ID NO：7832	0.53％	30
					13	966	SEQ ID NO：7833	0.44％	25
14	1387	SEQ ID NO：7834	0.44％	25
					15	2263	SEQ ID NO：7835	0.44％	25

16	2457	SEQ ID NO：7836	0.26％	15
					17	1057	SEQ ID NO：7837	0.22％	12.5
18	2562	SEQ ID NO：7838	0.22％	12.5
					19	74	SEQ ID NO：7839	0.17％	10
20	298	SEQ ID NO：7840	0.17％	10

HLA A3-9 mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	536	SEQ ID NO：7841	3.33％	405
2	986	SEQ ID NO：7842	2.46％	300
					3	805	SEQ ID NO：7843	1.64％	200
4	2345	SEQ ID NO：7844	1.48％	180
					5	2481	SEQ ID NO：7845	0.55％	67.5
6	204	SEQ ID NO：7846	0.49％	60
					7	895	SEQ ID NO：7847	0.44％	54
8	1512	SEQ ID NO：7848	0.44％	54
					9	2491	SEQ ID NO：7849	0.37％	45
10	436	SEQ ID NO：7850	0.32％	40
					11	917	SEQ ID NO：7851	0.32％	40
12	1176	SEQ ID NO：7852	0.32％	40
					13	1517	SEQ ID NO：7853	0.29％	36
14	466	SEQ ID NO：7854	0.24％	30
					15	1784	SEQ ID NO：7855	0.24％	30
16	2039	SEQ ID NO：7856	0.24％	30
					17	2124	SEQ ID NO：7857	0.24％	30
18	1049	SEQ ID NO：7858	0.22％	27
					19	2200	SEQ ID NO：7859	0.22％	27
20	2598	SEQ ID NO：7860	0.22％	27

HLA A3-10 mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	392	SEQ ID NO：7861	2.46％	300
2	2230	SEQ ID NO：7862	1.48％	180
					3	590	SEQ ID NO：7863	1.11％	135
4	697	SEQ ID NO：7864	1.11％	135
					5	919	SEQ ID NO：7865	0.74％	90
6	1354	SEQ ID NO：7866	0.74％	90
					7	1430	SEQ ID NO：7867	0.74％	90
8	2534	SEQ ID NO：7868	0.74％	90
					9	202	SEQ ID NO：7869	0.49％	60

10	488	SEQ ID NO：7870	0.49％	60
					11	922	SEQ ID NO：7871	0.49％	60
12	1735	SEQ ID NO：7872	0.49％	60
					13	2281	SEQ ID NO：7873	0.49％	60
14	1894	SEQ ID NO：7874	0.44％	54
					15	2552	SEQ ID NO：7875	0.44％	54
16	555	SEQ ID NO：7876	0.37％	45
					17	1134	SEQ ID NO：7877	0.37％	45
18	1149	SEQ ID NO：7878	0.29％	36
					19	283	SEQ ID NO：7879	0.24％	30
20	917	SEQ ID NO：7880	0.24％	30

HLA A24-9 mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	2375	SEQ ID NO：7881	36.07％	576
2	1751	SEQ ID NO：7882	28.93％	462
					3	195	SEQ ID NO：7883	25.05％	400
4	2306	SEQ ID NO：7884	21.04％	336
					5	806	SEQ ID NO：7885	20.66％	330
6	1252	SEQ ID NO：7886	18.78％	300
					7	160	SEQ ID NO：7887	15.03％	240
8	517	SEQ ID NO：7888	15.03％	240
					9	375	SEQ ID NO：7889	12.52％	200
10	1275	SEQ ID NO：7890	12.52％	200
					11	2175	SEQ ID NO：7891	12.52％	200
12	2207	SEQ ID NO：7892	12.52％	200
					13	2343	SEQ ID NO：7893	12.52％	200
14	443	SEQ ID NO：7894	11.27％	180
					15	668	SEQ ID NO：7895	7.51％	120
16	1825	SEQ ID NO：7896	6.88％	110
					17	1690	SEQ ID NO：7897	4.69％	75
18	159	SEQ ID NO：7898	3.75％	60
					19	2550	SEQ ID NO：7899	3.75％	60
20	1949	SEQ ID NO：7900	3.38％	54

HLA A24-10 mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	641	SEQ ID NO：7901	45.09％	720
2	809	SEQ ID NO：7902	24.80％	396
					3	1209	SEQ ID NO：7903	22.54％	360

4	216	SEQ ID NO：7904	18.03％	288
					5	159	SEQ ID NO：7905	15.03％	240
6	528	SEQ ID NO：7906	15.03％	240
					7	799	SEQ ID NO：7907	15.03％	240
8	1436	SEQ ID NO：7908	15.03％	240
					9	2219	SEQ ID NO：7909	15.03％	240
10	1065	SEQ ID NO：7910	13.77％	220
					11	1953	SEQ ID NO：7911	13.15％	210
12	1966	SEQ ID NO：7912	12.52％	200
					13	2600	SEQ ID NO：7913	12.52％	200
14	71	SEQ ID NO：7914	9.39％	150
					15	380	SEQ ID NO：7915	9.39％	150
16	1989	SEQ ID NO：7916	9.39％	150
					17	342	SEQ ID NO：7917	8.76％	140
18	1071	SEQ ID NO：7918	8.76％	140
					19	2570	SEQ ID NO：7919	6.88％	110
20	2550	SEQ ID NO：7920	6.26％	100

HLA A 0201-9 mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1632	SEQ ID NO：7921	0.09％	3607.31448
2	1640	SEQ ID NO：7922	0.04％	1748.2560912
					3	1776	SEQ ID NO：7923	0.03％	1492.58592
4	2512	SEQ ID NO：7924	0.03％	1434.16845
					5	1073	SEQ ID NO：7925	0.03％	1338.876
6	230	SEQ ID NO：7926	0.01％	685.78272
					7	1001	SEQ ID NO：7927	0.01％	559.8936
8	716	SEQ ID NO：7928	0.01％	558.27486
					9	2280	SEQ ID NO：7929	0.01％	511.19781048
10	590	SEQ ID NO：7930	0.01％	469.6692
					11	664	SEQ ID NO：7931	0.01％	442.076389524
12	1094	SEQ ID NO：7932	0.00％	382.536
					13	1735	SEQ ID NO：7933	0.00％	382.536
14	1625	SEQ ID NO：7934	0.00％	342.4606344
					15	1974	SEQ ID NO：7935	0.00％	336.885048
16	2382	SEQ ID NO：7936	0.00％	319.9392
					17	2417	SEQ ID NO：7937	0.00％	319.9392
18	744	SEQ ID NO：7938	0.00％	256.416670125
					19	108	SEQ ID NO：7939	0.00％	232.52724
20	390	SEQ ID NO：7940	0.00％	228.0411084

HLA A 0201-10 mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	2511	SEQ ID NO：7941	0.38％	15126.90795
2	1608	SEQ ID NO：7942	0.05％	2049.4656
					3	2572	SEQ ID NO：7943	0.04％	1879.5921264
4	255	SEQ ID NO：7944	0.03％	1566.6522795
					5	895	SEQ ID NO：7945	0.03％	1338.876
6	1171	SEQ ID NO：7946	0.02％	1107.960876
					7	1691	SEQ ID NO：7947	0.01％	782.95521024
8	20	SEQ ID NO：7948	0.01％	549.9372312
					9	1632	SEQ ID NO：7949	0.01％	479.041993296
10	2280	SEQ ID NO：7950	0.01％	472.418344576987
					11	1963	SEQ ID NO：7951	0.00％	358.73928
12	1955	SEQ ID NO：7952	0.00％	331.093464
					13	741	SEQ ID NO：7953	0.00％	318.652488
14	523	SEQ ID NO：7954	0.00％	278.7876
					15	1073	SEQ ID NO：7955	0.00％	266.6988828
16	2489	SEQ ID NO：7956	0.00％	243.432
					17	777	SEQ ID NO：7957	0.00％	218.5730664
18	1737	SEQ ID NO：7958	0.00％	218.0785572
					19	589	SEQ ID NO：7959	0.00％	210.538251
20	229	SEQ ID NO：7960	0.00％	205.230564

HLA A 1101-9 mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	2337	SEQ ID NO：7961	33.33％	12
2	2156	SEQ ID NO：7962	25％	9
					3	492	SEQ ID NO：7963	20％	7.2
4	18	SEQ ID NO：7964	16.66％	6
					5	332	SEQ ID NO：7965	16.66％	6
6	415	SEQ ID NO：7966	16.66％	6
					7	2479	SEQ ID NO：7967	16.66％	6
8	1495	SEQ ID NO：7968	11.11％	4
					9	2035	SEQ ID NO：7969	11.11％	4
10	1349	SEQ ID NO：7970	10％	3.6
					11	1194	SEQ ID NO：7971	8.33％	3
12	1648	SEQ ID NO：7972	8.33％	3
					13	96	SEQ ID NO：7973	6.66％	2.4
14	764	SEQ ID NO：7974	6.66％	2.4
					15	986	SEQ ID NO：7975	6.66％	2.4

16	2345	SEQ ID NO：7976	6.66％	2.4
					17	698	SEQ ID NO：7977	5.55％	2
18	1355	SEQ ID NO：7978	5.55％	2
					19	1987	SEQ ID NO：7979	5.55％	2
20	2085	SEQ ID NO：7980	5.55％	2

HLA A 1101-10 mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	2083	SEQ ID NO：7981	33.33％	12
2	2123	SEQ ID NO：7982	25％	9
					3	2147	SEQ ID NO：7983	16.66％	6
4	331	SEQ ID NO：7984	12.5％	4.5
					5	1035	SEQ ID NO：7985	11.11％	4
6	1064	SEQ ID NO：7986	11.11％	4
					7	2154	SEQ ID NO：7987	11.11％	4
8	1048	SEQ ID NO：7988	7.5％	2.7
					9	202	SEQ ID NO：7989	6.66％	2.4
10	721	SEQ ID NO：7990	6.66％	2.4
					11	2109	SEQ ID NO：7991	6.66％	2.4
12	2230	SEQ ID NO：7992	6.66％	2.4
					13	1306	SEQ ID NO：7993	5.55％	2
14	1622	SEQ ID NO：7994	5.55％	2
					15	1772	SEQ ID NO：7995	5.55％	2
16	1796	SEQ ID NO：7996	5.55％	2
					17	186	SEQ ID NO：7997	5％	1.8
18	414	SEQ ID NO：7998	5％	1.8
					19	697	SEQ ID NO：7999	5％	1.8
20	1175	SEQ ID NO：8000	5％	1.8

HLA B7-9 mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1447	SEQ ID NO：8001	14.81％	800
2	642	SEQ ID NO：8002	3.70％	200
					3	34	SEQ ID NO：8003	2.22％	120
4	186	SEQ ID NO：8004	1.48％	80
					5	244	SEQ ID NO：8005	1.48％	80
6	459	SEQ ID NO：8006	1.48％	80
					7	1475	SEQ ID NO：8007	1.48％	80
8	1867	SEQ ID NO：8008	1.48％	80
					9	2032	SEQ ID NO：8009	1.48％	80

10	2047	SEQ ID NO：8010	1.48％	80
					11	2335	SEQ ID NO：8011	1.48％	80
12	622	SEQ ID NO：8012	1.11％	60
					13	1375	SEQ ID NO：8013	1.11％	60
14	1617	SEQ ID NO：8014	0.92％	50
					15	1023	SEQ ID NO：8015	0.83％	45
16	286	SEQ ID NO：8016	0.74％	40
					17	490	SEQ ID NO：8017	0.74％	40
18	810	SEQ ID NO：8018	0.74％	40
					19	1420	SEQ ID NO：8019	0.74％	40
20	1854	SEQ ID NO：8020	0.74％	40

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1617	SEQ ID NO：8021	3.70％	200
2	752	SEQ ID NO：8022	2.22％	120
					3	1552	SEQ ID NO：8023	2.22％	120
4	154	SEQ ID NO：8024	1.48％	80
					5	165	SEQ ID NO：8025	1.48％	80
6	383	SEQ ID NO：8026	1.48％	80
					7	1501	SEQ ID NO：8027	1.48％	80
8	2093	SEQ ID NO：8028	1.48％	80
					9	2564	SEQ ID NO：8029	1.48％	80
10	622	SEQ ID NO：8030	1.11％	60
					11	1086	SEQ ID NO：8031	1.11％	60
12	1262	SEQ ID NO：8032	1.11％	60
					13	1556	SEQ ID NO：8033	1.11％	60
14	845	SEQ ID NO：8034	1％	54
					15	286	SEQ ID NO：8035	0.74％	40
16	490	SEQ ID NO：8036	0.74％	40
					17	552	SEQ ID NO：8037	0.74％	40
18	1858	SEQ ID NO：8038	0.74％	40
					19	2107	SEQ ID NO：8039	0.74％	40
20	2582	SEQ ID NO：8040	0.74％	40

The epi-position of table 16:SEQ ID NO:6042

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	846	SEQ ID NO：8041	2.22％	125

2	798	SEQ ID NO：8042	1.6％	90
					3	787	SEQ ID NO：8043	0.88％	50
4	1178	SEQ ID NO：8044	0.88％	50
					5	637	SEQ ID NO：8045	0.8％	45
6	557	SEQ ID NO：8046	0.44％	25
					7	1020	SEQ ID NO：8047	0.44％	25
8	282	SEQ ID NO：8048	0.32％	18
					9	1241	SEQ ID NO：8049	0.24％	13.5
10	466	SEQ ID NO：8050	0.22％	12.5
					11	727	SEQ ID NO：8051	0.2％	11.25
12	706	SEQ ID NO：8052	0.17％	10
					13	324	SEQ ID NO：8053	0.16％	9
14	752	SEQ ID NO：8054	0.16％	9
					15	54	SEQ ID NO：8055	0.13％	7.5
16	554	SEQ ID NO：8056	0.13％	7.5
					17	590	SEQ ID NO：8057	0.12％	6.75
18	569	SEQ ID NO：8058	0.08％	5
					19	613	SEQ ID NO：8059	0.08％	5
20	90	SEQ ID NO：8060	0.08％	4.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1241	SEQ ID NO：8061	4.8％	270
2	967	SEQ ID NO：8062	0.8％	45
					3	1010	SEQ ID NO：8063	0.48％	27
4	426	SEQ ID NO：8064	0.44％	25
					5	809	SEQ ID NO：8065	0.44％	25
6	1178	SEQ ID NO：8066	0.44％	25
					7	787	SEQ ID NO：8067	0.22％	12.5
8	958	SEQ ID NO：8068	0.22％	12.5
					9	727	SEQ ID NO：8069	0.2％	11.25
10	610	SEQ ID NO：8070	0.17％	10
					11	12	SEQ ID NO：8071	0.13％	7.5
12	1181	SEQ ID NO：8072	0.12％	6.75
					13	373	SEQ ID NO：8073	0.11％	6.25
14	602	SEQ ID NO：8074	0.11％	6.25
					15	20	SEQ ID NO：8075	0.04％	2.5
16	32	SEQ ID NO：8076	0.04％	2.5
					17	53	SEQ ID NO：8077	0.04％	2.5
18	400	SEQ ID NO：8078	0.04％	2.5
					19	557	SEQ ID NO：8079	0.04％	2.5

20

667

SEQ ID NO：8080

0.04％

2.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	768	SEQ ID NO：8081	0.82％	100
2	808	SEQ ID NO：8082	0.49％	60
					3	85	SEQ ID NO：8083	0.24％	30
4	663	SEQ ID NO：8084	0.24％	30
					5	1245	SEQ ID NO：8085	0.14％	18
6	288	SEQ ID NO：8086	0.09％	12
					7	50	SEQ ID NO：8087	0.08％	10
8	320	SEQ ID NO：8088	0.07％	9
					9	402	SEQ ID NO：8089	0.07％	9
10	798	SEQ ID NO：8090	0.07％	9
					11	902	SEQ ID NO：8091	0.06％	8.1
12	364	SEQ ID NO：8092	0.05％	6.75
					13	297	SEQ ID NO：8093	0.04％	6
14	992	SEQ ID NO：8094	0.04％	6
					15	38	SEQ ID NO：8095	0.03％	4.5
16	249	SEQ ID NO：8096	0.03％	4.5
					17	706	SEQ ID NO：8097	0.03％	4.05
18	1204	SEQ ID NO：8098	0.03％	4.05
					19	1178	SEQ ID NO：8099	0.03％	4
20	343	SEQ ID NO：8100	0.02％	3.6

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	255	SEQ ID NO：8101	1.48％	180
2	180	SEQ ID NO：8102	0.55％	67.5
					3	768	SEQ ID NO：8103	0.49％	60
4	1177	SEQ ID NO：8104	0.49％	60
					5	380	SEQ ID NO：8105	0.24％	30
6	100	SEQ ID NO：8106	0.18％	22.5
					7	786	SEQ ID NO：8107	0.16％	20
8	1217	SEQ ID NO：8108	0.16％	20
					9	207	SEQ ID NO：8109	0.14％	18
10	1183	SEQ ID NO：8110	0.14％	18
					11	38	SEQ ID NO：8111	0.09％	12
12	52	SEQ ID NO：8112	0.09％	12
					13	8	SEQ ID NO：8113	0.06％	8

14	679	SEQ ID NO：8114	0.06％	8
					15	73	SEQ ID NO：8115	0.05％	6.75
16	1204	SEQ ID NO：8116	0.05％	6.075
					17	50	SEQ ID NO：8117	0.04％	6
18	774	SEQ ID NO：8118	0.04％	6
					19	845	SEQ ID NO：8119	0.04％	6
20	214	SEQ ID NO：8120	0.04％	5.4

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1118	SEQ ID NO：8121	19.84％	316.8
2	51	SEQ ID NO：8122	18.78％	300
					3	161	SEQ ID NO：8123	18.78％	300
4	434	SEQ ID NO：8124	18.78％	300
					5	365	SEQ ID NO：8125	13.77％	220
6	736	SEQ ID NO：8126	12.52％	200
					7	620	SEQ ID NO：8127	7.51％	120
8	1068	SEQ ID NO：8128	7.51％	120
					9	817	SEQ ID NO：8129	3.75％	60
10	336	SEQ ID NO：8130	3.44％	55
					11	687	SEQ ID NO：8131	3.13％	50
12	254	SEQ ID NO：8132	2.34％	37.5
					13	627	SEQ ID NO：8133	1.87％	30
14	950	SEQ ID NO：8134	1.75％	28
					15	28	SEQ ID NO：8135	1.56％	25
16	408	SEQ ID NO：8136	1.56％	25
					17	159	SEQ ID NO：8137	1.31％	21
18	1166	SEQ ID NO：8138	1.26％	20.16
					19	45	SEQ ID NO：8139	1.25％	20
20	185	SEQ ID NO：8140	1.25％	20

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	438	SEQ ID NO：8141	27.55％	440
2	489	SEQ ID NO：8142	22.54％	360
					3	254	SEQ ID NO：8143	18.78％	300
4	354	SEQ ID NO：8144	11.27％	180
					5	406	SEQ ID NO：8145	11.27％	180
6	1047	SEQ ID NO：8146	11.27％	180
					7	473	SEQ ID NO：8147	7.51％	120

8	350	SEQ ID NO：8148	6.26％	100
					9	769	SEQ ID NO：8149	6.26％	100
10	193	SEQ ID NO：8150	5.63％	90
					11	479	SEQ ID NO：8151	3.13％	50
12	0	SEQ ID NO：8152	2.70％	43.2
					13	813	SEQ ID NO：8153	1.87％	30
14	739	SEQ ID NO：8154	1.50％	24
					15	782	SEQ ID NO：8155	1.50％	24
16	1186	SEQ ID NO：8156	1.31％	21
					17	910	SEQ ID NO：8157	1.05％	16.8
18	128	SEQ ID NO：8158	0.93％	15
					19	183	SEQ ID NO：8159	0.93％	15
20	1069	SEQ ID NO：8160	0.93％	15

HLA A0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	1041	SEQ ID NO：8161	0.01％	484.2379773
2	981	SEQ ID NO：8162	0.00％	382.536
					3	957	SEQ ID NO：8163	0.00％	342.4606344
4	896	SEQ ID NO：8164	0.00％	232.6931712
					5	1173	SEQ ID NO：8165	0.00％	201.447432
6	733	SEQ ID NO：8166	0.00％	171.86796
					7	410	SEQ ID NO：8167	0.00％	135.45252
8	786	SEQ ID NO：8168	0.00％	119.463012
					9	150	SEQ ID NO：8169	0.00％	102.17550222
10	1	SEQ ID NO：8170	0.00％	94.98737754
					11	595	SEQ ID NO：8171	0.00％	93.239424
12	1095	SEQ ID NO：8172	0.00％	89.41779
					13	1166	SEQ ID NO：8173	0.00％	87.58584
14	845	SEQ ID NO：8174	0.00％	79.642008
					15	734	SEQ ID NO：8175	0.00％	73.47672
16	802	SEQ ID NO：8176	0.00％	71.872056
					17	1213	SEQ ID NO：8177	0.00％	71.872056
18	105	SEQ ID NO：8178	0.00％	50.232
					19	939	SEQ ID NO：8179	0.00％	49.13352
20	130	SEQ ID NO：8180	0.00％	48.732354

HLA A0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	372	SEQ ID NO：8181	0.04％	1896.33528

2	410	SEQ ID NO：8182	0.02％	1134.00849744
					3	162	SEQ ID NO：8183	0.01％	685.3897512
4	1076	SEQ ID NO：8184	0.01％	640.90320525
					5	1196	SEQ ID NO：8185	0.01％	623.742666372
6	353	SEQ ID NO：8186	0.01％	446.7384576
					7	50	SEQ ID NO：8187	0.00％	375.97824
8	733	SEQ ID NO：8188	0.00％	271.863864
					9	130	SEQ ID NO：8189	0.00％	235.6873848
10	415	SEQ ID NO：8190	0.00％	185.679
					11	297	SEQ ID NO：8191	0.00％	177.496704
12	1	SEQ ID NO：8192	0.00％	152.42160582
					13	56	SEQ ID NO：8193	0.00％	110.013876
14	732	SEQ ID NO：8194	0.00％	101.0988
					15	6	SEQ ID NO：8195	0.00％	98.26704
16	261	SEQ ID NO：8196	0.00％	91.60164
					17	1040	SEQ ID NO：8197	0.00％	76.98537
18	928	SEQ ID NO：8198	0.00％	71.2908
					19	1188	SEQ ID NO：8199	0.00％	69.81282
20	1094	SEQ ID NO：8200	0.00％	52.5987

HLA A1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	402	SEQ ID NO：8201	25％	9
2	902	SEQ ID NO：8202	22.5％	8.1
					3	288	SEQ ID NO：8203	11.11％	4
4	85	SEQ ID NO：8204	6.66％	2.4
					5	706	SEQ ID NO：8205	6.66％	2.4
6	456	SEQ ID NO：8206	5.55％	2
					7	920	SEQ ID NO：8207	5.55％	2
8	535	SEQ ID NO：8208	5％	1.8
					9	364	SEQ ID NO：8209	3.33％	1.2
10	438	SEQ ID NO：8210	3.33％	1.2
					11	798	SEQ ID NO：8211	3.33％	1.2
12	808	SEQ ID NO：8212	3.33％	1.2
					13	937	SEQ ID NO：8213	3.33％	1.2
14	956	SEQ ID NO：8214	3.33％	1.2
					15	557	SEQ ID NO：8215	2.77％	1
16	1218	SEQ ID NO：8216	2.77％	1
					17	784	SEQ ID NO：8217	2.5％	0.9
18	249	SEQ ID NO：8218	2.22％	0.8
					19	768	SEQ ID NO：8219	2.22％	0.8

20

1178

SEQ ID NO：8220

2.22％

0.8

HLA A1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	38	SEQ ID NO：8221	13.33％	4.8
2	807	SEQ ID NO：8222	12.5％	4.5
					3	100	SEQ ID NO：8223	11.11％	4
4	380	SEQ ID NO：8224	11.11％	4
					5	767	SEQ ID NO：8225	10％	3.6
6	533	SEQ ID NO：8226	8.33％	3
					7	967	SEQ ID NO：8227	6.66％	2.4
8	919	SEQ ID NO：8228	5.55％	2
					9	305	SEQ ID NO：8229	5％	1.8
10	211	SEQ ID NO：8230	3.33％	1.2
					11	511	SEQ ID NO：8231	3.33％	1.2
12	1177	SEQ ID NO：8232	3.33％	1.2
					13	429	SEQ ID NO：8233	2.77％	1
14	758	SEQ ID NO：8234	2.77％	1
					15	797	SEQ ID NO：8235	2.5％	0.9
16	255	SEQ ID NO：8236	2.22％	0.8
					17	986	SEQ ID NO：8237	2.22％	0.8
18	1157	SEQ ID NO：8238	2.22％	0.8
					19	170	SEQ ID NO：8239	1.66％	0.6
20	893	SEQ ID NO：8240	1.66％	0.6

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	200	SEQ ID NO：8241	1.48％	80
2	1243	SEQ ID NO：8242	1.48％	80
					3	123	SEQ ID NO：8243	0.74％	40
4	248	SEQ ID NO：8244	0.66％	36
					5	1036	SEQ ID NO：8245	0.66％	36
6	494	SEQ ID NO：8246	0.37％	20
					7	495	SEQ ID NO：8247	0.37％	20
8	523	SEQ ID NO：8248	0.37％	20
					9	842	SEQ ID NO：8249	0.37％	20
10	932	SEQ ID NO：8250	0.37％	20
					11	274	SEQ ID NO：8251	0.33％	18
12	588	SEQ ID NO：8252	0.22％	12
					13	656	SEQ ID NO：8253	0.22％	12

14	657	SEQ ID NO：8254	0.22％	12
					15	767	SEQ ID NO：8255	0.22％	12
16	911	SEQ ID NO：8256	0.22％	12
					17	939	SEQ ID NO：8257	0.22％	12
18	1007	SEQ ID NO：8258	0.22％	12
					19	1170	SEQ ID NO：8259	0.22％	12
20	1206	SEQ ID NO：8260	0.22％	12

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	505	SEQ ID NO：8261	4.44％	240
2	312	SEQ ID NO：8262	3.70％	200
					3	141	SEQ ID NO：8263	1.11％	60
4	1006	SEQ ID NO：8264	0.66％	36
					5	411	SEQ ID NO：8265	0.44％	24
6	122	SEQ ID NO：8266	0.37％	20
					7	134	SEQ ID NO：8267	0.37％	20
8	184	SEQ ID NO：8268	0.37％	20
					9	367	SEQ ID NO：8269	0.37％	20
10	402	SEQ ID NO：8270	0.37％	20
					11	494	SEQ ID NO：8271	0.37％	20
12	560	SEQ ID NO：8272	0.37％	20
					13	626	SEQ ID NO：8273	0.37％	20
14	931	SEQ ID NO：8274	0.37％	20
					15	956	SEQ ID NO：8275	0.37％	20
16	1117	SEQ ID NO：8276	0.37％	20
					17	1169	SEQ ID NO：8277	0.37％	20
18	1196	SEQ ID NO：8278	0.37％	20
					19	247	SEQ ID NO：8279	0.22％	12
20	273	SEQ ID NO：8280	0.22％	12

The epi-position of table 17:SEQ ID NO:6043

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	168	SEQ ID NO：8281	0.2％	11.25
2	212	SEQ ID NO：8282	0.08％	4.5
					3	223	SEQ ID NO：8283	0.08％	4.5
4	104	SEQ ID NO：8284	0.04％	2.5
					5	170	SEQ ID NO：8285	0.04％	2.5

6	99	SEQ ID NO：8286	0.04％	2.25
					7	188	SEQ ID NO：8287	0.02％	1.35
8	180	SEQ ID NO：8288	0.02％	1.25
					9	219	SEQ ID NO：8289	0.02％	1.25
10	18	SEQ ID NO：8290	0.01％	1
					11	226	SEQ ID NO：8291	0.01％	1
12	98	SEQ ID NO：8292	0.01％	0.625
					13	151	SEQ ID NO：8293	0.01％	0.625
14	10	SEQ ID NO：8294	0.01％	0.6
					15	13	SEQ ID NO：8295	0.00％	0.5
16	32	SEQ ID NO：8296	0.00％	0.5
					17	70	SEQ ID NO：8297	0.00％	0.5
18	78	SEQ ID NO：8298	0.00％	0.5
					19	82	SEQ ID NO：8299	0.00％	0.5
20	145	SEQ ID NO：8300	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	99	SEQ ID NO：8301	0.8％	45
2	223	SEQ ID NO：8302	0.8％	45
					3	188	SEQ ID NO：8303	0.48％	27
4	206	SEQ ID NO：8304	0.2％	11.25
					5	253	SEQ ID NO：8305	0.17％	10
6	174	SEQ ID NO：8306	0.13％	7.5
					7	97	SEQ ID NO：8307	0.04％	2.5
8	257	SEQ ID NO：8308	0.04％	2.5
					9	179	SEQ ID NO：8309	0.04％	2.25
10	162	SEQ ID NO：8310	0.02％	1.25
					11	196	SEQ ID NO：8311	0.02％	1.25
12	219	SEQ ID NO：8312	0.02％	1.25
					13	18	SEQ ID NO：8313	0.01％	1
14	246	SEQ ID NO：8314	0.01％	1
					15	38	SEQ ID NO：8315	0.01％	0.75
16	33	SEQ ID NO：8316	0.00％	0.5
					17	69	SEQ ID NO：8317	0.00％	0.5
18	81	SEQ ID NO：8318	0.00％	0.5
					19	104	SEQ ID NO：8319	0.00％	0.5
20	116	SEQ ID NO：8320	0.00％	0.5

HLA A3-9mers
		Use the maximum possible of this molecule type to score	12150

Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	104	SEQ ID NO：8321	0.98％	120
2	123	SEQ ID NO：8322	0.74％	90
					3	82	SEQ ID NO：8323	0.44％	54
4	106	SEQ ID NO：8324	0.11％	13.5
					5	99	SEQ ID NO：8325	0.08％	10.8
6	127	SEQ ID NO：8326	0.08％	10
					7	71	SEQ ID NO：8327	0.07％	9
8	1	SEQ ID NO：8328	0.06％	8.1
					9	113	SEQ ID NO：8329	0.04％	6
10	84	SEQ ID NO：8330	0.03％	4.5
					11	109	SEQ ID NO：8331	0.03％	4.05
12	58	SEQ ID NO：8332	0.02％	3
					13	138	SEQ ID NO：8333	0.02％	3
14	44	SEQ ID NO：8334	0.02％	2.7
					15	81	SEQ ID NO：8335	0.02％	2.7
16	226	SEQ ID NO：8336	0.02％	2.7
					17	184	SEQ ID NO：8337	0.01％	1.8
18	102	SEQ ID NO：8338	0.01％	1.215
					19	39	SEQ ID NO：8339	0.00％	1.2
20	234	SEQ ID NO：8340	0.00％	0.9

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	99	SEQ ID NO：8341	1.33％	162
2	81	SEQ ID NO：8342	0.44％	54
					3	104	SEQ ID NO：8343	0.24％	30
4	51	SEQ ID NO：8344	0.16％	20
					5	122	SEQ ID NO：8345	0.11％	13.5
6	71	SEQ ID NO：8346	0.07％	9
					7	69	SEQ ID NO：8347	0.04％	6
8	223	SEQ ID NO：8348	0.04％	5.4
					9	84	SEQ ID NO：8349	0.03％	4.5
10	63	SEQ ID NO：8350	0.02％	3.6
					11	138	SEQ ID NO：8351	0.02％	3
12	201	SEQ ID NO：8352	0.01％	1.8
					13	44	SEQ ID NO：8353	0.01％	1.35
14	83	SEQ ID NO：8354	0.01％	1.35
					15	116	SEQ ID NO：8355	0.00％	1.2
16	46	SEQ ID NO：8356	0.00％	0.9
					17	183	SEQ ID NO：8357	0.00％	0.81

18	57	SEQ ID NO：8358	0.00％	0.6
					19	93	SEQ ID NO：8359	0.00％	0.6
20	113	SEQ ID NO：8360	0.00％	0.6

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	198	SEQ ID NO：8361	13.15％	210
2	105	SEQ ID NO：8362	9.39％	150
					3	210	SEQ ID NO：8363	4.69％	75
4	75	SEQ ID NO：8364	3.15％	50.4
					5	85	SEQ ID NO：8365	2.63％	42
6	205	SEQ ID NO：8366	2.10％	33.6
					7	77	SEQ ID NO：8367	1.87％	30
8	158	SEQ ID NO：8368	0.65％	10.5
					9	103	SEQ ID NO：8369	0.56％	9
10	227	SEQ ID NO：8370	0.55％	8.8704
					11	32	SEQ ID NO：8371	0.54％	8.64
12	74	SEQ ID NO：8372	0.50％	8
					13	131	SEQ ID NO：8373	0.50％	8
14	54	SEQ ID NO：8374	0.46％	7.5
					15	99	SEQ ID NO：8375	0.45％	7.2
16	44	SEQ ID NO：8376	0.37％	6
					17	62	SEQ ID NO：8377	0.37％	6
18	87	SEQ ID NO：8378	0.37％	6
					19	89	SEQ ID NO：8379	0.37％	6
20	154	SEQ ID NO：8380	0.37％	6

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	105	SEQ ID NO：8381	22.54％	360
2	204	SEQ ID NO：8382	17.53％	280
					3	209	SEQ ID NO：8383	3.13％	50
4	75	SEQ ID NO：8384	1.87％	30
					5	85	SEQ ID NO：8385	1.87％	30
6	77	SEQ ID NO：8386	1.12％	18
					7	74	SEQ ID NO：8387	0.84％	13.44
8	210	SEQ ID NO：8388	0.56％	9
					9	226	SEQ ID NO：8389	0.55％	8.8704
10	98	SEQ ID NO：8390	0.54％	8.64
					11	198	SEQ ID NO：8391	0.46％	7.5

12	67	SEQ ID NO：8392	0.45％	7.2
					13	152	SEQ ID NO：8393	0.43％	7
14	43	SEQ ID NO：8394	0.37％	6
					15	63	SEQ ID NO：8395	0.37％	6
16	72	SEQ ID NO：8396	0.37％	6
					17	89	SEQ ID NO：8397	0.37％	6
18	101	SEQ ID NO：8398	0.37％	6
					19	107	SEQ ID NO：8399	0.37％	6
20	111	SEQ ID NO：8400	0.37％	6

HLA A0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	138	SEQ ID NO：8401	0.21％	8532.082944
2	106	SEQ ID NO：8402	0.10％	3977.8497792
					3	44	SEQ ID NO：8403	0.03％	1243.078056
4	71	SEQ ID NO：8404	0.00％	348.872832
					5	234	SEQ ID NO：8405	0.00％	243.432
6	51	SEQ ID NO：8406	0.00％	130.26096
					7	109	SEQ ID NO：8407	0.00％	91.182672
8	81	SEQ ID NO：8408	0.00％	73.342584
					9	88	SEQ ID NO：8409	0.00％	70.386624
10	1	SEQ ID NO：8410	0.00％	65.32728732
					11	38	SEQ ID NO：8411	0.00％	47.876409
12	76	SEQ ID NO：8412	0.00％	36.8637882
					13	46	SEQ ID NO：8413	0.00％	30.889782
14	211	SEQ ID NO：8414	0.00％	21.616753941
					15	201	SEQ ID NO：8415	0.00％	19.657134
16	102	SEQ ID NO：8416	0.00％	18.4318941
					17	199	SEQ ID NO：8417	0.00％	16.496865
18	74	SEQ ID NO：8418	0.00％	15.783256167
					19	62	SEQ ID NO：8419	0.00％	13.9968225
20	99	SEQ ID NO：8420	0.00％	10.31851392

HLA A0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	78	SEQ ID NO：8421	0.01％	556.494246
2	138	SEQ ID NO：8422	0.01％	395.245972224
					3	84	SEQ ID NO：8423	0.00％	201.554244
4	71	SEQ ID NO：8424	0.00％	143.65707264
					5	44	SEQ ID NO：8425	0.00％	132.54624

6	76	SEQ ID NO：8426	0.00％	84.78671286
					7	8	SEQ ID NO：8427	0.00％	69.552
8	211	SEQ ID NO：8428	0.00％	52.7237901
					9	113	SEQ ID NO：8429	0.00％	47.99088
10	61	SEQ ID NO：8430	0.00％	37.4509575
					11	93	SEQ ID NO：8431	0.00％	31.24872
12	137	SEQ ID NO：8432	0.00％	31.1384304
					13	37	SEQ ID NO：8433	0.00％	27.531
14	55	SEQ ID NO：8434	0.00％	22.9153278
					15	98	SEQ ID NO：8435	0.00％	22.1063618985
16	108	SEQ ID NO：8436	0.00％	21.55457052
					17	63	SEQ ID NO：8437	0.00％	21.3624
18	45	SEQ ID NO：8438	0.00％	19.657134
					19	200	SEQ ID NO：8439	0.00％	19.657134
20	104	SEQ ID NO：8440	0.00％	13.87622016

HLA A1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	58	SEQ ID NO：8441	5.55％	2
2	125	SEQ ID NO：8442	1.66％	0.6
					3	226	SEQ ID NO：8443	1.66％	0.6
4	229	SEQ ID NO：8444	1.66％	0.6

HLA A1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	122	SEQ ID NO：8445	2.22％	0.8
2	228	SEQ ID NO：8446	2.22％	0.8

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	97	SEQ ID NO：8447	0.66％	36
2	86	SEQ ID NO：8448	0.37％	20
					3	37	SEQ ID NO：8449	0.33％	18
4	62	SEQ ID NO：8450	0.33％	18
					5	32	SEQ ID NO：8451	0.22％	12
6	102	SEQ ID NO：8452	0.22％	12
					7	227	SEQ ID NO：8453	0.22％	12
8	53	SEQ ID NO：8454	0.11％	6
					9	1	SEQ ID NO：8455	0.07％	4

10	44	SEQ ID NO：8456	0.07％	4
					11	56	SEQ ID NO：8457	0.07％	4
12	64	SEQ ID NO：8458	0.07％	4
					13	74	SEQ ID NO：8459	0.07％	4
14	76	SEQ ID NO：8460	0.07％	4
					15	87	SEQ ID NO：8461	0.07％	4
16	106	SEQ ID NO：8462	0.07％	4
					17	131	SEQ ID NO：8463	0.07％	4
18	23	SEQ ID NO：8464	0.03％	2
					19	157	SEQ ID NO：8465	0.03％	2
20	166	SEQ ID NO：8466	0.03％	2

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	119	SEQ ID NO：8467	3.33％	180
2	264	SEQ ID NO：8468	1.48％	80
					3	98	SEQ ID NO：8469	0.66％	36
4	27	SEQ ID NO：8470	0.37％	20
					5	86	SEQ ID NO：8471	0.37％	20
6	31	SEQ ID NO：8472	0.22％	12
					7	63	SEQ ID NO：8473	0.22％	12
8	96	SEQ ID NO：8474	0.22％	12
					9	101	SEQ ID NO：8475	0.22％	12
10	226	SEQ ID NO：8476	0.22％	12
					11	157	SEQ ID NO：8477	0.14％	8
12	176	SEQ ID NO：8478	0.14％	8
					13	238	SEQ ID NO：8479	0.14％	8
14	36	SEQ ID NO：8480	0.11％	6
					15	53	SEQ ID NO：8481	0.11％	6
16	61	SEQ ID NO：8482	0.11％	6
					17	3	SEQ ID NO：8483	0.07％	4
18	40	SEQ ID NO：8484	0.07％	4
					19	55	SEQ ID NO：8485	0.07％	4
20	74	SEQ ID NO：8486	0.07％	4

The epi-position of table 18:SEQ ID NO:6044

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	69	SEQ ID NO：8487	0.04％	2.5

2	89	SEQ ID NO：8488	0.02％	1.5
					3	141	SEQ ID NO：8489	0.01％	1
4	113	SEQ ID NO：8490	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	21	SEQ ID NO：8491	0.02％	1.5
2	88	SEQ ID NO：8492	0.02％	1.5
					3	8	SEQ ID NO：8493	0.02％	1.25
4	31	SEQ ID NO：8494	0.00％	0.5
					5	112	SEQ ID NO：8495	0.00％	0.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	60	SEQ ID NO：8496	1.23％	150
2	77	SEQ ID NO：8497	1.11％	135
					3	141	SEQ ID NO：8498	0.49％	60
4	95	SEQ ID NO：8499	0.32％	40
					5	128	SEQ ID NO：8500	0.08％	10
6	113	SEQ ID NO：8501	0.04％	6
					7	69	SEQ ID NO：8502	0.01％	2
8	22	SEQ ID NO：8503	0.01％	1.8
					9	42	SEQ ID NO：8504	0.01％	1.8
10	78	SEQ ID NO：8505	0.00％	1.2
					11	32	SEQ ID NO：8506	0.00％	1
12	54	SEQ ID NO：8507	0.00％	0.9
					13	74	SEQ ID NO：8508	0.00％	0.9
14	28	SEQ ID NO：8509	0.00％	0.6
					15	36	SEQ ID NO：8510	0.00％	0.6
16	48	SEQ ID NO：8511	0.00％	0.6
					17	118	SEQ ID NO：8512	0.00％	0.6
18	4	SEQ ID NO：8513	0.00％	0.5

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	94	SEQ ID NO：8514	0.49％	60
2	48	SEQ ID NO：8515	0.16％	20
					3	128	SEQ ID NO：8516	0.16％	20
4	60	SEQ ID NO：8517	0.12％	15

5	127	SEQ ID NO：8518	0.12％	15
					6	25	SEQ ID NO：8519	0.04％	6
7	95	SEQ ID NO：8520	0.04％	6
					8	141	SEQ ID NO：8521	0.04％	6
9	41	SEQ ID NO：8522	0.04％	5.4
					10	77	SEQ ID NO：8523	0.04％	5.4
11	116	SEQ ID NO：8524	0.04％	5.4
					12	91	SEQ ID NO：8525	0.03％	4
13	4	SEQ ID NO：8526	0.01％	2
					14	112	SEQ ID NO：8527	0.01％	1.8
15	113	SEQ ID NO：8528	0.01％	1.35
					16	12	SEQ ID NO：8529	0.00％	1.2
17	31	SEQ ID NO：8530	0.00％	1
					18	32	SEQ ID NO：8531	0.00％	1
19	15	SEQ ID NO：8532	0.00％	0.9
					20	27	SEQ ID NO：8533	0.00％	0.9

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	61	SEQ ID NO：8534	14.46％	231
2	16	SEQ ID NO：8535	3.13％	50
					3	120	SEQ ID NO：8536	1.87％	30
4	41	SEQ ID NO：8537	0.60％	9.6
					5	71	SEQ ID NO：8538	0.45％	7.2
6	21	SEQ ID NO：8539	0.37％	6
					7	53	SEQ ID NO：8540	0.37％	6
8	65	SEQ ID NO：8541	0.37％	6
					9	121	SEQ ID NO：8542	0.37％	6
10	74	SEQ ID NO：8543	0.36％	5.76
					11	20	SEQ ID NO：8544	0.35％	5.6
12	79	SEQ ID NO：8545	0.35％	5.6
					13	105	SEQ ID NO：8546	0.33％	5.28
14	48	SEQ ID NO：8547	0.30％	4.8
					15	88	SEQ ID NO：8548	0.30％	4.8
16	106	SEQ ID NO：8549	0.30％	4.8
					17	37	SEQ ID NO：8550	0.27％	4.4
18	70	SEQ ID NO：8551	0.27％	4.4
					19	18	SEQ ID NO：8552	0.25％	4
20	57	SEQ ID NO：8553	0.22％	3.6

HLA A24-10mers

Use the maximum possible of this molecule type to score				1596.672
					Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
1	120	SEQ ID NO：8554	1.87％	30
					2	73	SEQ ID NO：8555	0.54％	8.64
3	19	SEQ ID NO：8556	0.52％	8.4
					4	78	SEQ ID NO：8557	0.52％	8.4
5	104	SEQ ID NO：8558	0.49％	7.92
					6	61	SEQ ID NO：8559	0.46％	7.5
7	47	SEQ ID NO：8560	0.45％	7.2
					8	36	SEQ ID NO：8561	0.41％	6.6
9	52	SEQ ID NO：8562	0.37％	6
					10	64	SEQ ID NO：8563	0.30％	4.8
11	70	SEQ ID NO：8564	0.30％	4.8
					12	105	SEQ ID NO：8565	0.30％	4.8
13	123	SEQ ID NO：8566	0.30％	4.8
					14	69	SEQ ID NO：8567	0.27％	4.4
15	20	SEQ ID NO：8568	0.25％	4
					16	66	SEQ ID NO：8569	0.25％	4
17	83	SEQ ID NO：8570	0.25％	4
					18	86	SEQ ID NO：8571	0.25％	4
19	101	SEQ ID NO：8572	0.25％	4
					20	119	SEQ ID NO：8573	0.25％	4

HLA A0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	62	SEQ ID NO：8574	0.00％	136.1646
2	85	SEQ ID NO：8575	0.00％	69.6969
					3	47	SEQ ID NO：8576	0.00％	60.153786
4	121	SEQ ID NO：8577	0.00％	52.5182736
					5	74	SEQ ID NO：8578	0.00％	49.13352
6	23	SEQ ID NO：8579	0.00％	21.99582
					7	78	SEQ ID NO：8580	0.00％	19.42488
8	114	SEQ ID NO：8581	0.00％	14.6900655
					9	4	SEQ ID NO：8582	0.00％	11.304684
10	79	SEQ ID NO：8583	0.00％	8.4687081
					11	122	SEQ ID NO：8584	0.00％	6.0996
12	100	SEQ ID NO：8585	0.00％	5.382
					13	105	SEQ ID NO：8586	0.00％	4.981593
14	25	SEQ ID NO：8587	0.00％	4.968
					15	115	SEQ ID NO：8588	0.00％	4.966482
16	24	SEQ ID NO：8589	0.00％	4.4815221585

17	111	SEQ ID NO：8590	0.00％	4.128201
					18	94	SEQ ID NO：8591	0.00％	3.67632
19	34	SEQ ID NO：8592	0.00％	3.47553
					20	12	SEQ ID NO：8593	0.00％	3.30993

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	77	SEQ ID NO：8594	0.00％	147.97188
2	62	SEQ ID NO：8595	0.00％	143.59176
					3	113	SEQ ID NO：8596	0.00％	106.83684
4	78	SEQ ID NO：8597	0.00％	83.526984
					5	86	SEQ ID NO：8598	0.00％	83.526984
6	74	SEQ ID NO：8599	0.00％	69.552
					7	121	SEQ ID NO：8600	0.00％	61.06776
8	12	SEQ ID NO：8601	0.00％	50.232
					9	44	SEQ ID NO：8602	0.00％	26.082
10	4	SEQ ID NO：8603	0.00％	18.3816
					11	0	SEQ ID NO：8604	0.00％	17.38386
12	72	SEQ ID NO：8605	0.00％	17.1396
					13	22	SEQ ID NO：8606	0.00％	16.21914
14	122	SEQ ID NO：8607	0.00％	14.02908
					15	64	SEQ ID NO：8608	0.00％	11.161854
16	46	SEQ ID NO：8609	0.00％	10.34586
					17	54	SEQ ID NO：8610	0.00％	8.846145
18	47	SEQ ID NO：8611	0.00％	7.575080337
					19	131	SEQ ID NO：8612	0.00％	7.452
20	114	SEQ ID NO：8613	0.00％	6.735366

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	69	SEQ ID NO：8614	5.55％	2
2	22	SEQ ID NO：8615	5％	1.8
					3	77	SEQ ID NO：8616	5％	1.8
4	141	SEQ ID NO：8617	3.33％	1.2
					5	60	SEQ ID NO：8618	2.22％	0.8
6	95	SEQ ID NO：8619	2.22％	0.8
					7	36	SEQ ID NO：8620	1.66％	0.6

HLA A 1101-10mers
		Use the maximum possible of this molecule type to score	36

Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	41	SEQ ID NO：8621	3.33％	1.2
2	68	SEQ ID NO：8622	3.33％	1.2
					3	94	SEQ ID NO：8623	3.33％	1.2
4	31	SEQ ID NO：8624	2.77％	1
					5	127	SEQ ID NO：8625	2.5％	0.9

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	48	SEQ ID NO：8626	0.74％	40
2	20	SEQ ID NO：8627	0.37％	20
					3	121	SEQ ID NO：8628	0.33％	18
4	18	SEQ ID NO：8629	0.07％	4
					5	21	SEQ ID NO：8630	0.07％	4
6	37	SEQ ID NO：8631	0.07％	4
					7	41	SEQ ID NO：8632	0.07％	4
8	53	SEQ ID NO：8633	0.07％	4
					9	65	SEQ ID NO：8634	0.07％	4
10	70	SEQ ID NO：8635	0.07％	4
					11	71	SEQ ID NO：8636	0.07％	4
12	74	SEQ ID NO：8637	0.07％	4
					13	79	SEQ ID NO：8638	0.07％	4
14	88	SEQ ID NO：8639	0.07％	4
					15	105	SEQ ID NO：8640	0.07％	4
16	106	SEQ ID NO：8641	0.07％	4
					17	124	SEQ ID NO：8642	0.07％	4
18	1	SEQ ID NO：8643	0.03％	2
					19	120	SEQ ID NO：8644	0.03％	1.8
20	11	SEQ ID NO：8645	0.02％	1.2

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	66	SEQ ID NO：8646	1.48％	80
2	123	SEQ ID NO：8647	0.74％	40
					3	20	SEQ ID NO：8648	0.37％	20
4	64	SEQ ID NO：8649	0.22％	12
					5	119	SEQ ID NO：8650	0.11％	6
6	54	SEQ ID NO：8651	0.09％	5
					7	19	SEQ ID NO：8652	0.07％	4
8	36	SEQ ID NO：8653	0.07％	4

9	47	SEQ ID NO：8654	0.07％	4
					10	52	SEQ ID NO：8655	0.07％	4
11	69	SEQ ID NO：8656	0.07％	4
					12	70	SEQ ID NO：8657	0.07％	4
13	73	SEQ ID NO：8658	0.07％	4
					14	78	SEQ ID NO：8659	0.07％	4
15	83	SEQ ID NO：8660	0.07％	4
					16	86	SEQ ID NO：8661	0.07％	4
17	101	SEQ ID NO：8662	0.07％	4
					18	104	SEQ ID NO：8663	0.07％	4
19	105	SEQ ID NO：8664	0.07％	4
					20	15	SEQ ID NO：8665	0.03％	2

The epi-position of table 19:SEQ ID NO:6045

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	4	SEQ ID NO：8666	0.02％	1.35
2	66	SEQ ID NO：8667	0.02％	1.35
					3	33	SEQ ID NO：8668	0.02％	1.25
4	44	SEQ ID NO：8669	0.01％	1
					5	50	SEQ ID NO：8670	0.01％	1
6	14	SEQ ID NO：8671	0.01％	0.75
					7	48	SEQ ID NO：8672	0.01％	0.75
8	11	SEQ ID NO：8673	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	4	SEQ ID NO：8674	0.12％	6.75
2	66	SEQID NO：8675	0.12％	6.75
					3	10	SEQ ID NO：8676	0.00％	0.5
4	28	SEQ ID NO：8677	0.00％	0.5
					5	32	SEQ ID NO：8678	0.00％	0.5
6	47	SEQ ID NO：8679	0.00％	0.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	17	SEQ ID NO：8680	0.24％	30
2	44	SEQ ID NO：8681	0.07％	9

3	19	SEQ ID NO：8682	0.06％	8.1
					4	50	SEQ ID NO：8683	0.04％	5.4
5	29	SEQ ID NO：8684	0.03％	4
					6	52	SEQ ID NO：8685	0.02％	3.24
7	54	SEQ ID NO：8686	0.02％	3
					8	11	SEQ ID NO：8687	0.01％	1.8
9	37	SEQ ID NO：8688	0.01％	1.8
					10	25	SEQ ID NO：8689	0.01％	1.35
11	10	SEQ ID NO：8690	0.00％	0.9
					12	16	SEQ ID NO：8691	0.00％	0.9
13	35	SEQ ID NO：8692	0.00％	0.6

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	49	SEQ ID NO：8693	0.44％	54
2	17	SEQ ID NO：8694	0.22％	27
					3	10	SEQ ID NO：8695	0.14％	18
4	16	SEQ ID NO：8696	0.07％	9
					5	32	SEQ ID NO：8697	0.04％	6
6	19	SEQ ID NO：8698	0.01％	1.8
					7	29	SEQ ID NO：8699	0.00％	1.2
8	23	SEQ ID NO：8700	0.00％	0.9
					9	26	SEQ ID NO：8701	0.00％	0.9

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	18	SEQ ID NO：8702	1.87％	30
2	24	SEQ ID NO：8703	0.65％	10.5
					3	9	SEQ ID NO：8704	0.52％	8.4
4	12	SEQ ID NO：8705	0.52％	8.4
					5	28	SEQ ID NO：8706	0.52％	8.4
6	42	SEQ ID NO：8707	0.52％	8.4
					7	57	SEQ ID NO：8708	0.52％	8.4
8	66	SEQ ID NO：8709	0.52％	8.4
					9	55	SEQ ID NO：8710	0.51％	8.25
10	0	SEQ ID NO：8711	0.48％	7.7
					11	22	SEQ ID NO：8712	0.45％	7.2
12	10	SEQ ID NO：8713	0.37％	6
					13	25	SEQ ID NO：8714	0.37％	6
14	30	SEQ ID NO：8715	0.37％	6

15	19	SEQ ID NO：8716	0.35％	5.6
					16	40	SEQ ID NO：8717	0.31％	5
17	3	SEQ ID NO：8718	0.30％	4.8
					18	65	SEQ ID NO：8719	0.30％	4.8
19	14	SEQ ID NO：8720	0.27％	4.32
					20	56	SEQ ID NO：8721	0.25％	4

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	55	SEQ ID NO：8722	18.78％	300
2	18	SEQ ID NO：8723	2.63％	42
					3	21	SEQ ID NO：8724	2.25％	36
4	2	SEQ ID NO：8725	1.87％	30
					5	24	SEQ ID NO：8726	1.87％	30
6	11	SEQ ID NO：8727	0.52％	8.4
					7	40	SEQ ID NO：8728	0.52％	8.4
8	65	SEQ ID NO：8729	0.42％	6.72
					9	9	SEQ ID NO：8730	0.37％	6
10	8	SEQ ID NO：8731	0.35％	5.6
					11	27	SEQ ID NO：8732	0.35％	5.6
12	41	SEQ ID NO：8733	0.35％	5.6
					13	57	SEQ ID NO：8734	0.31％	5
14	17	SEQ ID NO：8735	0.25％	4
					15	29	SEQ ID NO：8736	0.25％	4
16	64	SEQ ID NO：8737	0.25％	4
					17	16	SEQ ID NO：8738	0.22％	3.6
18	10	SEQ ID NO：8739	0.18％	3
					19	13	SEQ ID NO：8740	0.18％	2.88
20	23	SEQ ID NO：8741	0.08％	1.4

HLA A 0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	19	SEQ ID NO：8742	0.03％	1310.8823136
2	15	SEQ ID NO：8743	0.02％	1082.4143022
					3	16	SEQ ID NO：8744	0.02％	1040.33238624
4	49	SEQ ID NO：8745	0.00％	382.536
					5	25	SEQ ID NO：8746	0.00％	342.863529264
6	56	SEQ ID NO：8747	0.00％	63.28397376
					7	12	SEQ ID NO：8748	0.00％	40.19736105
8	10	SEQ ID NO：8749	0.00％	21.3624

9	22	SEQ ID NO：8750	0.00％	19.7762418
					10	26	SEQ ID NO：8751	0.00％	12.6684
11	20	SEQ ID NO：8752	0.00％	11.544666
					12	37	SEQ ID NO：8753	0.00％	10.4328
13	32	SEQ ID NO：8754	0.00％	8.4456
					14	23	SEQ ID NO：8755	0.00％	6.2888049
15	47	SEQ ID NO：8756	0.00％	6.0858
					16	3	SEQ ID NO：8757	0.00％	4.582929078
17	18	SEQ ID NO：8758	0.00％	4.4855150505
					18	28	SEQ ID NO：8759	0.00％	4.2923589
19	62	SEQ ID NO：8760	0.00％	2.88098391
					20	27	SEQ ID NO：8761	0.00％	1.699677

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	17	SEQ ID NO：8762	0.16％	6459.14167272
2	19	SEQ ID NO：8763	0.01％	607.88448
					3	25	SEQ ID NO：8764	0.00％	126.83304
4	11	SEQ ID NO：8765	0.00％	63.16728165
					5	15	SEQ ID NO：8766	0.00％	53.54651988
6	37	SEQ ID NO：8767	0.00％	28.51632
					7	14	SEQ ID NO：8768	0.00％	21.8247414
8	29	SEQ ID NO：8769	0.00％	21.3624
					9	26	SEQ ID NO：8770	0.00％	19.42488
10	3	SEQ ID NO：8771	0.00％	17.2167282
					11	48	SEQ ID NO：8772	0.00％	15.7068219
12	12	SEQ ID NO：8773	0.00％	9.8581266
					13	27	SEQ ID NO：8774	0.00％	7.3086111
14	39	SEQ ID NO：8775	0.00％	7.10976
					15	23	SEQ ID NO：8776	0.00％	5.7419523
16	22	SEQ ID NO：8777	0.00％	4.599126
					17	45	SEQ ID NO：8778	0.00％	2.5495155
18	31	SEQ ID NO：8779	0.00％	2.52747
					19	52	SEQ ID NO：8780	0.00％	2.383605
20	20	SEQ ID NO：8781	0.00％	2.332847151

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	44	SEQ ID NO：8782	3.33％	1.2

HLA A 1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	3	SEQ ID NO：8783	0.37％	20
2	12	SEQ ID NO：8784	0.37％	20
					3	22	SEQ ID NO：8785	0.37％	20
4	56	SEQ ID NO：8786	0.37％	20
					5	30	SEQ ID NO：8787	0.22％	12
6	9	SEQ ID NO：8788	0.07％	4
					7	10	SEQ ID NO：8789	0.07％	4
8	19	SEQ ID NO：8790	0.07％	4
					9	25	SEQ ID NO：8791	0.07％	4
10	28	SEQ ID NO：8792	0.07％	4
					11	42	SEQ ID NO：8793	0.07％	4
12	65	SEQ ID NO：8794	0.07％	4
					13	35	SEQ ID NO：8795	0.05％	3
14	66	SEQ ID NO：8796	0.02％	1.2
					15	15	SEQ ID NO：8797	0.01％	1
16	47	SEQ ID NO：8798	0.01％	1
					17	20	SEQ ID NO：8799	0.01％	0.6
18	23	SEQ ID NO：8800	0.00％	0.5
					19	27	SEQ ID NO：8801	0.00％	0.5

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	27	SEQ ID NO：8802	0.37％	20
2	8	SEQ ID NO：8803	0.07％	4
					3	9	SEQ ID NO：8804	0.07％	4
4	11	SEQ ID NO：8805	0.07％	4
					5	17	SEQ ID NO：8806	0.07％	4
6	29	SEQ ID NO：8807	0.07％	4
					7	41	SEQ ID NO：8808	0.07％	4
8	52	SEQ ID NO：8809	0.07％	4
					9	64	SEQ ID NO：8810	0.07％	4
10	65	SEQ ID NO：8811	0.07％	4
					11	3	SEQ ID NO：8812	0.03％	2
12	23	SEQ ID NO：8813	0.03％	2

13	21	SEQ ID NO：8814	0.02％	1.2
					14	15	SEQ ID NO：8815	0.01％	1
15	35	SEQ ID NO：8816	0.01％	0.6
					16	39	SEQ ID NO：8817	0.01％	0.6
17	12	SEQ ID NO：8818	0.00％	0.5
					18	22	SEQ ID NO：8819	0.00％	0.5
19	45	SEQ ID NO：8820	0.00％	0.5

The epi-position of table 20:SEQ ID NO:6046

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	186	SEQ ID NO：8821	2.22％	125
2	156	SEQ ID NO：8822	0.88％	50
					3	14	SEQ ID NO：8823	0.08％	4.5
4	0	SEQ ID NO：8824	0.04％	2.5
					5	29	SEQ ID NO：8825	0.04％	2.5
6	85	SEQ ID NO：8826	0.04％	2.5
					7	168	SEQ ID NO：8827	0.04％	2.5
8	133	SEQ ID NO：8828	0.02％	1.35
					9	111	SEQ ID NO：8829	0.02％	1.125
10	61	SEQ ID NO：8830	0.01％	1
					11	7	SEQ ID NO：8831	0.01％	0.9
12	131	SEQ ID NO：8832	0.01％	0.9
					13	211	SEQ ID NO：8833	0.01％	0.625
14	4	SEQ ID NO：8834	0.00％	0.5
					15	43	SEQ ID NO：8835	0.00％	0.5
16	95	SEQ ID NO：8836	0.00％	0.5
					17	136	SEQ ID NO：8837	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	133	SEQ ID NO：8838	0.04％	2.7
2	84	SEQ ID NO：8839	0.04％	2.5
					3	167	SEQ ID NO：8840	0.04％	2.5
4	186	SEQ ID NO：8841	0.04％	2.5
					5	131	SEQ ID NO：8842	0.04％	2.25
6	14	SEQ ID NO：8843	0.03％	1.8
					7	205	SEQ ID NO：8844	0.02％	1.25
8	111	SEQ ID NO：8845	0.02％	1.125

9	60	SEQ ID NO：8846	0.01％	1
					10	188	SEQ ID NO：8847	0.01％	0.75
11	211	SEQ ID NO：8848	0.01％	0.625
					12	26	SEQ ID NO：8849	0.00％	0.5
13	94	SEQ ID NO：8850	0.00％	0.5
					14	135	SEQ ID NO：8851	0.00％	0.5
15	168	SEQ ID NO：8852	0.00％	0.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	43	SEQ ID NO：8853	0.24％	30
2	90	SEQ ID NO：8854	0.14％	18
					3	148	SEQ ID NO：8855	0.09％	12
4	4	SEQ ID NO：8856	0.05％	6.75
					5	24	SEQ ID NO：8857	0.04％	6
6	19	SEQ ID NO：8858	0.04％	5.4
					7	136	SEQ ID NO：8859	0.04％	5.4
8	54	SEQ ID NO：8860	0.03％	4.5
					9	32	SEQ ID NO：8861	0.03％	4
10	14	SEQ ID NO：8862	0.02％	3.6
					11	59	SEQ ID NO：8863	0.02％	3.6
12	88	SEQ ID NO：8864	0.02％	3
					13	87	SEQ ID NO：8865	0.02％	2.7
14	29	SEQ ID NO：8866	0.01％	L 8
					15	48	SEQ ID NO：8867	0.01％	1.8
16	115	SEQ ID NO：8868	0.01％	1.8
					17	186	SEQ ID NO：8869	0.01％	1.8
18	106	SEQ ID NO：8870	0.01％	1.5
					19	53	SEQ ID NO：8871	0.01％	1.35
20	173	SEQ ID NO：8872	0.00％	1.2

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	24	SEQ ID NO：8873	0.22％	27
2	54	SEQ ID NO：8874	0.18％	22.5
					3	135	SEQ ID NO：8875	0.08％	10.8
4	51	SEQ ID NO：8876	0.07％	9
					5	13	SEQ ID NO：8877	0.06％	8.1
6	26	SEQ ID NO：8878	0.04％	6
					7	31	SEQ ID NO：8879	0.04％	6

8	90	SEQ ID NO：8880	0.04％	6
					9	43	SEQ ID NO：8881	0.03％	4.5
10	19	SEQ ID NO：8882	0.03％	4.05
					11	169	SEQ ID NO：8883	0.02％	3
12	87	SEQ ID NO：8884	0.02％	2.7
					13	84	SEQ ID NO：8885	0.01％	1.8
14	88	SEQ ID NO：8886	0.01％	1.8
					15	94	SEQ ID NO：8887	0.01％	1.8
16	64	SEQ ID NO：8888	0.00％	1.2
					17	131	SEQ ID NO：8889	0.00％	1.2
18	99	SEQ ID NO：8890	0.00％	1
					19	53	SEQ ID NO：8891	0.00％	0.9
20	85	SEQ ID NO：8892	0.00％	0.9

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	196	SEQ ID NO：8893	27.55％	440
2	44	SEQ ID NO：8894	18.78％	300
					3	36	SEQ ID NO：8895	12.52％	200
4	92	SEQ ID NO：8896	12.52％	200
					5	109	SEQ ID NO：8897	2.70％	43.2
6	25	SEQ ID NO：8898	1.87％	30
					7	93	SEQ ID NO：8899	1.12％	18
8	12	SEQ ID NO：8900	0.75％	12
					9	123	SEQ ID NO：8901	0.70％	11.2
10	7	SEQ ID NO：8902	0.64％	10.368
					11	17	SEQ ID NO：8903	0.52％	8.4
12	139	SEQ ID NO：8904	0.52％	8.4
					13	193	SEQ ID NO：8905	0.46％	7.5
14	6	SEQ ID NO：8906	0.45％	7.2
					15	19	SEQ ID NO：8907	0.45％	7.2
16	110	SEQ ID NO：8908	0.45％	7.2
					17	114	SEQ ID NO：8909	0.45％	7.2
18	210	SEQ ID NO：8910	0.45％	7.2
					19	46	SEQ ID NO：8911	0.42％	6.72
20	52	SEQ ID NO：8912	0.37％	6

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	92	SEQ ID NO：8913	7.51％	120

2	42	SEQ ID NO：8914	2.63％	42
					3	109	SEQ ID NO：8915	2.25％	36
4	23	SEQ ID NO：8916	1.87％	30
					5	34	SEQ ID NO：8917	0.75％	12
6	6	SEQ ID NO：8918	0.64％	10.368
					7	45	SEQ ID NO：8919	0.63％	10.08
8	196	SEQ ID NO：8920	0.62％	10
					9	44	SEQ ID NO：8921	0.56％	9
10	40	SEQ ID NO：8922	0.55％	8.8
					11	62	SEQ ID NO：8923	0.46％	7.5
12	193	SEQ ID NO：8924	0.46％	7.5
					13	18	SEQ ID NO：8925	0.45％	7.2
14	113	SEQ ID NO：8926	0.45％	7.2
					15	56	SEQ ID NO：8927	0.37％	6
16	176	SEQ ID NO：8928	0.37％	6
					17	16	SEQ ID NO：8929	0.35％	5.6
18	138	SEQ ID NO：8930	0.35％	5.6
					19	127	SEQ ID NO：8931	0.33％	5.28
20	36	SEQ ID NO：8932	0.31％	5

HLA A 0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	13	SEQ ID NO：8933	0.04％	1793.676528
2	87	SEQ ID NO：8934	0.03％	1415.3832
					3	24	SEQ ID NO：8935	0.01％	618.0996816
4	19	SEQ ID NO：8936	0.00％	223.23708
					5	12	SEQ ID NO：8937	0.00％	210.36400875
6	51	SEQ ID NO：8938	0.00％	198.30859992
					7	53	SEQ ID NO：8939	0.00％	194.477328
8	88	SEQ ID NO：8940	0.00％	180.58536756
					9	106	SEQ ID NO：8941	0.00％	169.74828
10	54	SEQ ID NO：8942	0.00％	70.09848
					11	59	SEQ ID NO：8943	0.00％	43.42032
12	94	SEQ ID NO：8944	0.00％	41.792058
					13	20	SEQ ID NO：8945	0.00％	37.46088108
14	63	SEQ ID NO：8946	0.00％	35.73520902
					15	22	SEQ ID NO：8947	0.00％	20.5916435109
16	47	SEQ ID NO：8948	0.00％	12.233222865
					17	66	SEQ ID NO：8949	0.00％	12.2199
18	56	SEQ ID NO：8950	0.00％	11.486706
					19	67	SEQ ID NO：8951	0.00％	6.416172

20

117

SEQ ID NO：8952

0.00％

5.827464

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	43	SEQ ID NO：8953	0.10％	3977.8497792
2	24	SEQ ID NO：8954	0.02％	836.2525104
					3	51	SEQ ID NO：8955	0.02％	815.616432
4	49	SEQ ID NO：8956	0.01％	660.3245145
					5	19	SEQ ID NO：8957	0.00％	251.837856
6	59	SEQ ID NO：8958	0.00％	159.9696
					7	12	SEQ ID NO：8959	0.00％	155.245377
8	45	SEQ ID NO：8960	0.00％	141.1974531
					9	21	SEQ ID NO：8961	0.00％	117.22672269
10	53	SEQ ID NO：8962	0.00％	84.55536
					11	87	SEQ ID NO：8963	0.00％	65.5671672
12	13	SEQ ID NO：8964	0.00％	64.88888616
					13	153	SEQ ID NO：8965	0.00％	49.13352
14	178	SEQ ID NO：8966	0.00％	26.082
					15	18	SEQ ID NO：8967	0.00％	24.802259691
16	116	SEQ ID NO：8968	0.00％	21.5616168
					17	65	SEQ ID NO：8969	0.00％	20.77383
18	86	SEQ ID NO：8970	0.00％	15.7068219
					19	27	SEQ ID NO：8971	0.00％	12.3159135
20	46	SEQ ID NO：8972	0.00％	11.45624789925

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	4	SEQ ID NO：8973	12.5％	4.5
2	136	SEQ ID NO：8974	3.33％	1.2
					3	156	SEQ ID NO：8975	3.33％	1.2
4	140	SEQ ID NO：8976	1.66％	0.6

HLA A 1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	169	SEQ ID NO：8977	5.55％	2
2	94	SEQ ID NO：8978	3.33％	1.2

HLA B7-9mers
		Use the maximum possible of this molecule type to score	5400

Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	146	SEQ ID NO：8979	0.74％	40
2	154	SEQ ID NO：8980	0.74％	40
					3	80	SEQ ID NO：8981	0.66％	36
4	139	SEQ ID NO：8982	0.33％	18
					5	83	SEQ ID NO：8983	0.22％	12
6	209	SEQ ID NO：8984	0.22％	12
					7	7	SEQ ID NO：8985	0.11％	6
8	3	SEQ ID NO：8986	0.07％	4
					9	6	SEQ ID NO：8987	0.07％	4
10	12	SEQ ID NO：8988	0.07％	4
					11	19	SEQ ID NO：8989	0.07％	4
12	24	SEQ ID NO：8990	0.07％	4
					13	38	SEQ ID NO：8991	0.07％	4
14	46	SEQ ID NO：8992	0.07％	4
					15	56	SEQ ID NO：8993	0.07％	4
16	110	SEQ ID NO：8994	0.07％	4
					17	114	SEQ ID NO：8995	0.07％	4
18	123	SEQ ID NO：8996	0.07％	4
					19	129	SEQ ID NO：8997	0.07％	4
20	166	SEQ ID NO：8998	0.07％	4

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	56	SEQ ID NO：8999	1.48％	80
2	40	SEQ ID NO：9000	0.74％	40
					3	127	SEQ ID NO：9001	0.74％	40
4	170	SEQ ID NO：9002	0.74％	40
					5	140	SEQ ID NO：9003	0.27％	15
6	35	SEQ ID NO：9004	0.22％	12
					7	79	SEQ ID NO：9005	0.22％	12
8	82	SEQ ID NO：9006	0.22％	12
					9	208	SEQ ID NO：9007	0.22％	12
10	209	SEQ ID NO：9008	0.22％	12
					11	80	SEQ ID NO：9009	0.16％	9
12	129	SEQ ID NO：9010	0.14％	8
					13	138	SEQ ID NO：9011	0.11％	6
14	73	SEQ ID NO：9012	0.09％	5
					15	2	SEQ ID NO：9013	0.07％	4
16	5	SEQ ID NO：9014	0.07％	4
					17	6	SEQ ID NO：9015	0.07％	4

18	16	SEQ ID NO：9016	0.07％	4
					19	18	SEQ ID NO：9017	0.07％	4
20	24	SEQ ID NO：9018	0.07％	4

The epi-position of table 21:SEQ ID NO:6047

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	53	SEQ ID NO：9019	2％	112.5
2	10	SEQ ID NO：9020	0.08％	4.5
					3	33	SEQ ID NO：9021	0.02％	1.5
4	3	SEQ ID NO：9022	0.00％	0.5
					5	27	SEQ ID NO：9023	0.00％	0.5
6	29	SEQ ID NO：9024	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	10	SEQ ID NO：9025	0.8％	45
2	52	SEQ ID NO：9026	0.2％	11.25
					3	50	SEQ ID NO：9027	0.04％	2.5
4	32	SEQ ID NO：9028	0.02％	1.5
					5	48	SEQ ID NO：9029	0.02％	1.35
6	27	SEQ ID NO：9030	0.00％	0.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	38	SEQ ID NO：9031	1.85％	225
2	17	SEQ ID NO：9032	0.02％	3.6
					3	2	SEQ ID NO：9033	0.02％	2.7
4	37	SEQ ID NO：9034	0.01％	1.8
					5	27	SEQ ID NO：9035	0.01％	1.35
6	13	SEQ ID NO：9036	0.00％	0.675
					7	14	SEQ ID NO：9037	0.00％	0.6

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	13	SEQ ID NO：9038	0.04％	6
2	37	SEQ ID NO：9039	0.01％	2.025

32	SEQ ID NO：9040	0.00％	0.9
				419	SEQ ID NO：9041	0.00％	0.675
516	SEQ ID NO：9042	0.00％	0.54

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	20	SEQ ID NO：9043	1.25％	20
2	6	SEQ ID NO：9044	0.52％	8.4
					3	5	SEQ ID NO：9045	0.51％	8.25
4	35	SEQ ID NO：9046	0.36％	5.76
					5	31	SEQ ID NO：9047	0.35％	5.6
6	43	SEQ ID NO：9048	0.27％	4.4
					7	13	SEQ ID NO：9049	0.26％	4.2
8	32	SEQ ID NO：9050	0.21％	3.36
					9	2	SEQ ID NO：9051	0.11％	1.8
10	9	SEQ ID NO：9052	0.10％	1.68
					11	8	SEQ ID NO：9053	0.09％	1.5
12	15	SEQ ID NO：9054	0.09％	1.5
					13	23	SEQ ID NO：9055	0.09％	1.5
14	27	SEQ ID NO：9056	0.08％	1.4
					15	24	SEQ ID NO：9057	0.07％	1.2
16	7	SEQ ID NO：9058	0.06％	1
					17	17	SEQ ID NO：9059	0.06％	1
18	10	SEQ ID NO：9060	0.05％	0.9
					19	39	SEQ ID NO：9061	0.04％	0.792
20	47	SEQ ID NO：9062	0.04％	0.792

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	5	SEQ ID NO：9063	2.63％	42
2	34	SEQ ID NO：9064	0.54％	8.64
					3	30	SEQ ID NO：9065	0.52％	8.4
4	19	SEQ ID NO：9066	0.50％	8
					5	50	SEQ ID NO：9067	0.33％	5.28
6	12	SEQ ID NO：9068	0.26％	4.2
					7	31	SEQ ID NO：9069	0.21％	3.36
8	26	SEQ ID NO：9070	0.15％	2.52
					9	8	SEQ ID NO：9071	0.13％	2.1
10	22	SEQ ID NO：9072	0.12％	2
					11	23	SEQ ID NO：9073	0.11％	1.8

12	6	SEQ ID NO：9074	0.09％	1.5
					13	14	SEQ ID NO：9075	0.09％	1.5
14	16	SEQ ID NO：9076	0.09％	1.5
					15	7	SEQ ID NO：9077	0.06％	1
16	48	SEQ ID NO：9078	0.04％	0.75
					17	0	SEQ ID NO：9079	0.04％	0.72
18	9	SEQ ID NO：9080	0.04％	0.72
					19	47	SEQ ID NO：9081	0.04％	0.66
20	39	SEQ ID NO：9082	0.03％	0.6

HLA A 0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	15	SEQ ID NO：9083	0.00％	14.1442686
2	27	SEQ ID NO：9084	0.00％	9.598176
					3	22	SEQ ID NO：9085	0.00％	9.5634
4	9	SEQ ID NO：9086	0.00％	5.546246013
					5	2	SEQ ID NO：9087	0.00％	5.526462816
6	24	SEQ ID NO：9088	0.00％	4.88163753
					7	17	SEQ ID NO：9089	0.00％	3.699285408
8	31	SEQ ID NO：9090	0.00％	2.29699206
					9	6	SEQ ID NO：9091	0.00％	2.0016040674
10	7	SEQ ID NO：9092	0.00％	0.91287
					11	49	SEQ ID NO：9093	0.00％	0.71805678
12	16	SEQ ID NO：9094	0.00％	0.6694257042
					13	12	SEQ ID NO：9095	0.00％	0.6539828625

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	16	SEQ ID NO：9096	0.00％	34.28765802
2	19	SEQ ID NO：9097	0.00％	18.9368775
					3	14	SEQ ID NO：9098	0.00％	14.1442686
4	27	SEQ ID NO：9099	0.00％	11.406528
					5	26	SEQ ID NO：9100	0.00％	10.9304361558
6	34	SEQ ID NO：9101	0.00％	5.580927
					7	6	SEQ ID NO：9102	0.00％	4.865742
8	9	SEQ ID NO：9103	0.00％	2.64106953
					9	50	SEQ ID NO：9104	0.00％	2.6275752
10	30	SEQ ID NO：9105	0.00％	2.29699206
					11	7	SEQ ID NO：9106	0.00％	0.86083641
12	42	SEQ ID NO：9107	0.00％	0.7049592

13	22	SEQ ID NO：9108	0.00％	0.6628440357
					14	2	SEQ ID NO：9109	0.00％	0.6530644656

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	37	SEQ ID NO：9110	15％	5.4
2	38	SEQ ID NO：9111	2.22％	0.8

HLA A 1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	37	SEQ ID NO：9112	7.5％	2.7

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	35	SEQ ID NO：9113	3.70％	200
2	17	SEQ ID NO：9114	0.11％	6
					3	6	SEQ ID NO：9115	0.07％	4
4	20	SEQ ID NO：9116	0.07％	4
					5	31	SEQ ID NO：9117	0.07％	4
6	43	SEQ ID NO：9118	0.07％	4
					7	7	SEQ ID NO：9119	0.03％	2
8	23	SEQ ID NO：9120	0.02％	1.2
					9	24	SEQ ID NO：9121	0.02％	1.2
10	10	SEQ ID NO：9122	0.01％	0.9

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	35	SEQ ID NO：9123	0.09％	5
2	19	SEQ ID NO：9124	0.07％	4
					3	30	SEQ ID NO：9125	0.07％	4
4	34	SEQ ID NO：9126	0.07％	4
					5	7	SEQ ID NO：9127	0.03％	2
6	16	SEQ ID NO：9128	0.03％	1.8
					7	23	SEQ ID NO：9129	0.02％	1.2
8	50	SEQ ID NO：9130	0.02％	1.2
					9	9	SEQ ID NO：9131	0.01％	1

The epi-position of table 22:SEQ ID NO:6048

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	66	SEQ ID NO：9132	0.44％	25
2	80	SEQ ID NO：9133	0.08％	5
					3	93	SEQ ID NO：9134	0.04％	2.7
4	11	SEQ ID NO：9135	0.04％	2.5
					5	89	SEQ ID NO：9136	0.04％	2.25
6	48	SEQ ID NO：9137	0.01％	1
					7	3	SEQ ID NO：9138	0.00％	0.5
8	9	SEQ ID NO：9139	0.00％	0.5
					9	56	SEQ ID NO：9140	0.00％	0.5
10	101	SEQ ID NO：9141	0.00％	0.5
					11	106	SEQ ID NO：9142	0.00％	0.5
12	110	SEQ ID NO：9143	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	30	SEQ ID NO：9144	0.4％	22.5
2	88	SEQ ID NO：9145	0.12％	6.75
					3	48	SEQ ID NO：9146	0.04％	2.5
4	55	SEQ ID NO：9147	0.02％	1.25
					5	13	SEQ ID NO：9148	0.01％	0.9
6	79	SEQ ID NO：9149	0.01％	0.75
					7	93	SEQ ID NO：9150	0.01％	0.675
8	2	SEQ ID NO：9151	0.00％	0.5
					9	8	SEQ ID NO：9152	0.00％	0.5
10	65	SEQ ID NO：9153	0.00％	0.5
					11	66	SEQ ID NO：9154	0.00％	0.5
12	80	SEQ ID NO：9155	0.00％	0.5
					13	105	SEQ ID NO：9156	0.00％	0.5
14	109	SEQ ID NO：9157	0.00％	0.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	109	SEQ ID NO：9158	0.74％	90
2	3	SEQ ID N0：9159	0.24％	30
					3	111	SEQ ID NO：9160	0.12％	15
4	106	SEQ ID NO：9161	0.07％	9
					5	95	SEQ ID NO：9162	0.05％	6.075

6	101	SEQ ID NO：9163	0.04％	6
					7	110	SEQ ID NO：9164	0.02％	3.6
8	84	SEQ ID NO：9165	0.02％	3
					9	80	SEQ ID NO：9166	0.02％	2.7
10	37	SEQ ID NO：9167	0.01％	2.25
					11	9	SEQ ID NO：9168	0.01％	2
12	54	SEQ ID NO：9169	0.01％	2
					13	99	SEQ ID NO：9170	0.01％	1.35
14	1	SEQ ID NO：9171	0.01％	1.215
					15	11	SEQ ID NO：9172	0.00％	0.9
16	15	SEQ ID NO：9173	0.00％	0.9
					17	69	SEQ ID NO：9174	0.00％	0.6
18	5	SEQ ID NO：9175	0.00％	0.54
					19	103	SEQ ID NO：9176	0.00％	0.54

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	75	SEQ ID NO：9177	0.49％	60
2	109	SEQ ID NO：9178	0.29％	36
					3	22	SEQ ID NO：9179	0.14％	18
4	15	SEQ ID NO：9180	0.04％	6
					5	110	SEQ ID NO：9181	0.01％	2.25
6	95	SEQ ID NO：9182	0.01％	1.8
					7	101	SEQ ID NO：9183	0.01％	1.35
8	43	SEQ ID NO：9184	0.00％	1
					9	2	SEQ ID NO：9185	0.00％	0.9
10	5	SEQ ID NO：9186	0.00％	0.9
					11	7	SEQ ID NO：9187	0.00％	0.9
12	107	SEQ ID NO：9188	0.00％	0.9
					13	102	SEQ ID NO：9189	0.00％	0.81
14	3	SEQ ID NO：9190	0.00％	0.75
					15	8	SEQ ID NO：9191	0.00％	0.6
16	103	SEQ ID NO：9192	0.00％	0.54

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	88	SEQ ID NO：9193	1.66％	26.6112
2	77	SEQ ID NO：9194	0.77％	12.32
					3	18	SEQ ID NO：9195	0.56％	9
4	108	SEQ ID NO：9196	0.56％	9

5	92	SEQ ID NO：9197	0.54％	8.64
					6	96	SEQ ID NO：9198	0.54％	8.64
7	73	SEQ ID N0：9199	0.46％	7.5
					8	40	SEQ ID NO：9200	0.45％	7.2
9	104	SEQ ID NO：9201	0.42％	6.72
					10	8	SEQ ID NO：9202	0.41％	6.6
11	21	SEQ ID NO：9203	0.37％	6
					12	102	SEQ ID NO：9204	0.37％	6
13	22	SEQ ID NO：9205	0.25％	4
					14	68	SEQ ID NO：9206	0.25％	4
15	106	SEQ ID NO：9207	0.22％	3.6
					16	1	SEQ ID NO：9208	0.18％	3
17	79	SEQ ID NO：9209	0.18％	3
					18	93	SEQ ID NO：9210	0.18％	3
19	101	SEQ ID NO：9211	0.18	3
					20	37	SEQ ID NO：9212	0.15％	2.4

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	100	SEQ ID NO：9213	0.93％	15
2	18	SEQ ID NO：9214	0.78％	12.6
					3	98	SEQ ID NO：9215	0.52％	8.4
4	73	SEQ ID NO：9216	0.46％	7.5
					5	91	SEQ ID NO：9217	0.45％	7.2
6	103	SEQ ID NO：9218	0.42％	6.72
					7	7	SEQ ID NO：9219	0.41％	6.6
8	21	SEQ ID NO：9220	0.37％	6
					9	46	SEQ ID NO：9221	0.37％	6
10	93	SEQ ID NO：9222	0.37％	6
					11	96	SEQ ID NO：9223	0.37％	6
12	101	SEQ ID NO：9224	0.37％	6
					13	77	SEQ ID NO：9225	0.25％	4
14	92	SEQ ID NO：9226	0.22％	3.6
					15	105	SEQ ID NO：9227	0.22％	3.6
16	2	SEQ ID NO：9228	0.18％	3
					17	53	SEQ ID NO：9229	0.18％	3
18	36	SEQ ID NO：9230	0.12％	2
					19	55	SEQ ID NO：9231	0.12％	2
20	102	SEQ ID NO：9232	0.11％	1.8

HLA A 0201-9mers

Use the maximum possible of this molecule type to score				3925227.1
					Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
1	84	SEQ ID NO：9233	0.01％	441.342216
					2	102	SEQ ID NO：9234	0.00％	63.16728165
3	107	SEQ ID NO：9235	0.00％	51.882640425
					4	1	SEQ ID NO：9236	0.00％	43.8816609
5	95	SEQ ID NO：9237	0.00％	33.40165248
					6	2	SEQ ID NO：9238	0.00％	24.66305226
7	92	SEQ ID NO：9239	0.00％	22.64458905
					8	103	SEQ ID NO：9240	0.00％	20.70206586
9	47	SEQ ID NO：9241	0.00％	11.175953184
					10	94	SEQ ID NO：9242	0.00％	8.452983
11	15	SEQ ID NO：9243	0.00％	8.1793152
					12	8	SEQ ID NO：9244	0.00％	4.993461
13	5	SEQ ID NO：9245	0.00％	4.57284528
					14	99	SEQ ID NO：9246	0.00％	3.999468528
15	105	SEQ ID NO：9247	0.00％	2.231322
					16	20	SEQ ID NO：9248	0.00％	1.3524
17	62	SEQ ID NO：9249	0.00％	0.8631693
					18	6	SEQ ID NO：9250	0.00％	0.824619
19	57	SEQ ID NO：9251	0.00％	0.72105
					20	58	SEQ ID NO：9252	0.00％	0.7147572

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	101	SEQ ID NO：9253	0.03％	1243.078056
2	3	SEQ ID NO：9254	0.01％	592.944462
					3	106	SEQ ID NO：9255	0.00％	94.2678
4	5	SEQ ID NO：9256	0.00％	43.42032
					5	107	SEQ ID NO：9257	0.00％	33.30332334
6	102	SEQ ID NO：9258	0.00％	32.53181778
					7	54	SEQ ID NO：9259	0.00％	27.324
8	7	SEQ ID NO：9260	0.00％	21.3624
					9	1	SEQ ID NO：9261	0.00％	13.723479
10	95	SEQ ID N0：9262	0.00％	13.00344192
					11	94	SEQ ID NO：9263	0.00％	10.01276388
12	99	SEQ ID NO：9264	0.00％	5.6615328
					13	39	SEQ ID NO：9265	0.00％	3.6304212
14	111	SEQ ID NO：9266	0.00％	2.53368
					15	103	SEQ ID NO：9267	0.00％	2.475394803
16	14	SEQ ID NO：9268	0.00％	2.4519012

17	19	SEQ ID NO：9269	0.00％	2.07604992
					18	29	SEQ ID NO：9270	0.00％	1.8179154
19	57	SEQ ID NO：9271	0.00％	1.52076
					20	47	SEQ ID NO：9272	0.00％	1.27712376

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	80	SEQ ID NO：9273	3.33％	1.2
2	69	SEQ ID NO：9274	1.66％	0.6
					3	109	SEQ ID NO：9275	1.66％	0.6

HLA A 1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	22	SEQ ID NO：9276	11.11％	4

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	22	SEQ ID NO：9277	3.70％	200
2	77	SEQ ID NO：9278	2.22％	120
					3	104	SEQ ID NO：9279	0.22％	12
4	40	SEQ ID NO：9280	0.11％	6
					5	8	SEQ ID NO：9281	0.07％	4
6	21	SEQ ID NO：9282	0.07％	4
					7	68	SEQ ID NO：9283	0.07％	4
8	92	SEQ ID NO：9284	0.07％	4
					9	102	SEQ ID NO：9285	0.07％	4
10	46	SEQ ID NO：9286	0.03％	2
					11	98	SEQ ID NO：9287	0.03％	2
12	103	SEQ ID NO：9288	0.03％	2
					13	88	SEQ ID NO：9289	0.02％	1.2
14	105	SEQ ID NO：9290	0.01％	0.9
					15	43	SEQ ID NO：9291	0.01％	0.6
16	79	SEQ ID NO：9292	0.01％	0.6
					17	95	SEQ ID NO：9293	0.01％	0.6
18	107	SEQ ID NO：9294	0.00％	0.5

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score

1	46	SEQ ID NO：9295	1.48％	80
					2	98	SEQ ID NO：9296	1.48％	80
3	91	SEQ ID NO：9297	0.37％	20
					4	103	SEQ ID NO：9298	0.37％	20
5	7	SEQ ID NO：9299	0.07％	4
					6	21	SEQ ID NO：9300	0.07％	4
7	101	SEQ ID NO：9301	0.07％	4
					8	107	SEQ ID NO：9302	0.03％	2
9	67	SEQ ID NO：9303	0.02％	1.2
					10	93	SEQ ID NO：9304	0.02％	1.2
11	69	SEQ ID NO：9305	0.01％	1
					12	39	SEQ ID NO：9306	0.01％	0.6
13	77	SEQ ID NO：9307	0.01％	0.6
					14	22	SEQ ID NO：9308	0.00％	0.5

The epi-position of table 23:SEQ ID NO:6049

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	0	SEQ ID NO：9309	0.2％	11.25
2	35	SEQ ID NO：9310	0.01％	0.9
					3	4	SEQ ID NO：9311	0.00％	0.5
4	5	SEQ ID NO：9312	0.00％	0.5
					5	10	SEQ ID NO：9313	0.00％	0.5
6	19	SEQ ID NO：9314	0.00％	0.5
					7	21	SEQ ID NO：9315	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	0	SEQ ID NO：9316	0.2％	11.25
2	5	SEQ ID NO：9317	0.04％	2.5
					3	33	SEQ ID NO：9318	0.02％	1.5
4	3	SEQ ID NO：9319	0.02％	1.25
					5	9	SEQ ID NO：9320	0.00％	0.5
6	18	SEQ ID NO：9321	0.00％	0.5
					7	20	SEQ ID NO：9322	0.00％	0.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score

1	4	SEQ ID NO：9323	0.14％	18
					2	16	SEQ ID NO：9324	0.11％	13.5
3	23	SEQ ID NO：9325	0.06％	8.1
					4	18	SEQ ID NO：9326	0.03％	4.05
5	21	SEQ ID NO：9327	0.01％	2.025
					6	9	SEQ ID NO：9328	0.01％	1.8
7	15	SEQ ID NO：9329	0.01％	1.8
					8	25	SEQ ID NO：9330	0.01％	1.8
9	12	SEQ ID NO：9331	0.00％	0.9
					10	19	SEQ ID NO：9332	0.00％	0.9
11	20	SEQ ID NO：9333	0.00％	0.9
					12	2	SEQ ID NO：9334	0.00％	0.81
13	22	SEQ ID NO：9335	0.00％	0.81
					14	10	SEQ ID NO：9336	0.00％	0.6

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	20	SEQ ID NO：9337	0.16％	20.25
2	9	SEQ ID NO：9338	0.09％	12
					3	16	SEQ ID NO：9339	0.07％	9
4	18	SEQ ID NO：9340	0.07％	9
					5	22	SEQ ID NO：9341	0.06％	8.1
6	4	SEQ ID NO：9342	0.03％	4.05
					7	15	SEQ ID NO：9343	0.03％	4.05
8	12	SEQ ID NO：9344	0.02％	3.6
					9	3	SEQ ID NO：9345	0.00％	0.9
10	33	SEQ ID NO：9346	0.00％	0.6
					11	2	SEQ ID NO：9347	0.00％	0.54
12	24	SEQ ID NO：9348	0.00％	0.54

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	8	SEQ ID NO：9349	18.78％	300
2	11	SEQ ID NO：9350	1.87％	30
					3	28	SEQ ID NO：9351	1.50％	24
4	7	SEQ ID NO：9352	0.75％	12
					5	17	SEQ ID NO：9353	0.56％	9
6	14	SEQ ID NO：9354	0.46％	7.5
					7	23	SEQ ID NO：9355	0.37％	6
8	13	SEQ ID NO：9356	0.36％	5.76

9	2	SEQ ID NO：9357	0.35％	5.6
					10	16	SEQ ID NO：9358	0.35％	5.6
11	9	SEQ ID NO：9359	0.30％	4.8
					12	21	SEQ ID NO：9360	0.26％	4.2
13	5	SEQ ID NO：9361	0.25％	4
					14	4	SEQ ID NO：9362	0.22％	3.6
15	0	SEQ ID NO：9363	0.18％	3
					16	19	SEQ ID NO：9364	0.18％	3
17	10	SEQ ID NO：9365	0.15％	2.4
					18	18	SEQ ID NO：9366	0.13％	2.1
19	25	SEQ ID NO：9367	0.06％	1.1
					20	15	SEQ ID NO：9368	0.05％	0.9

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	8	SEQ ID NO：9369	22.54％	360
2	7	SEQ ID NO：9370	1.25％	20
					3	17	SEQ ID NO：9371	0.65％	10.5
4	15	SEQ ID NO：9372	0.52％	8.4
					5	4	SEQ ID NO：9373	0.45％	7.2
6	22	SEQ ID NO：9374	0.37％	6
					7	12	SEQ ID NO：9375	0.36％	5.76
8	27	SEQ ID NO：9376	0.30％	4.8
					9	14	SEQ ID NO：9377	0.28％	4.5
10	20	SEQ ID NO：9378	0.26％	4.2
					11	10	SEQ ID NO：9379	0.25％	4
12	3	SEQ ID NO：9380	0.18％	3
					13	18	SEQ ID NO：9381	0.18％	3
14	9	SEQ ID NO：9382	0.15％	2.4
					15	24	SEQ ID NO：9383	0.10％	1.65
16	16	SEQ ID NO：9384	0.07％	1.2
					17	13	SEQ ID NO：9385	0.06％	1
18	11	SEQ ID NO：9386	0.05％	0.9
					19	1	SEQ ID NO：9387	0.05％	0.84

HLA A 0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	12	SEQ ID NO：9388	0.10％	4267.988928
2	23	SEQ ID NO：9389	0.03％	1360.69088544
					3	9	SEQ ID NO：9390	0.01％	569.948832

4	16	SEQ ID NO：9391	0.00％	309.0498408
					5	15	SEQ ID NO：9392	0.00％	79.73570448
6	2	SEQ ID NO：9393	0.00％	51.109542
					7	18	SEQ ID NO：9394	0.00％	45.25539984
8	25	SEQ ID NO：9395	0.00％	34.28765802
					9	22	SEQ ID NO：9396	0.00％	26.532116325
10	5	SEQ ID NO：9397	0.00％	25.26691266
					11	21	SEQ ID NO：9398	0.00％	4.72873208445
12	11	SEQ ID NO：9399	0.00％	2.638538265
					13	8	SEQ ID NO：9400	0.00％	2.4274552038
14	4	SEQ ID NO：9401	0.00％	1.7415324
					15	20	SEQ ID NO：9402	0.00％	1.6025526
16	13	SEQ ID NO：9403	0.00％	1.453803297
					17	35	SEQ ID NO：9404	0.00％	1.36878336
18	3	SEQ ID NO：9405	0.00％	0.824619
					19	33	SEQ ID NO：9406	0.00％	0.513774

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	22	SEQ ID NO：9407	0.09％	3636.068421648
2	4	SEQ ID NO：9408	0.02％	1107.960876
					3	15	SEQ ID NO：9409	0.02％	836.2525104
4	16	SEQ ID NO：9410	0.00％	150.9313176
					5	12	SEQ ID NO：9411	0.00％	76.55002416
6	1	SEQ ID NO：9412	0.00％	49.0273014
					7	10	SEQ ID NO：9413	0.00％	42.1638414747
8	20	SEQ ID NO：9414	0.00％	9.29480508
					9	24	SEQ ID NO：9415	0.00％	9.2669346
10	13	SEQ ID NO：9416	0.00％	7.96581954
					11	21	SEQ ID NO：9417	0.00％	5.051306761875
12	5	SEQ ID NO：9418	0.00％	2.6941464
					13	11	SEQ ID NO：9419	0.00％	2.3839914
14	34	SEQ ID NO：9420	0.00％	1.465422
					15	2	SEQ ID NO：9421	0.00％	0.70794
16	9	SEQ ID NO：9422	0.00％	0.6513048
					17	19	SEQID NO：9423	0.00％	0.51882640425

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score

HLA A 1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	33	SEQ ID NO：9424	1.66％	0.6

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	13	SEQ ID NO：9425	0.22％	12
2	2	SEQ ID NO：9426	0.07％	4
					3	9	SEQ ID NO：9427	0.07％	4
4	16	SEQ ID NO：9428	0.07％	4
					5	23	SEQ ID NO：9429	0.07％	4
6	5	SEQ ID NO：9430	0.02％	1.2
					7	15	SEQ ID NO：9431	0.01％	1

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	4	SEQ ID NO：9432	0.07％	4
2	10	SEQ ID NO：9433	0.07％	4
					3	12	SEQ ID NO：9434	0.07％	4
4	15	SEQ ID NO：9435	0.07％	4
					5	22	SEQ ID NO：9436	0.07％	4
6	13	SEQ ID NO：9437	0.02％	1.2

The epi-position of table 24:SEQ ID NO:6050

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	47	SEQ ID NO：9438	0.01％	0.75
2	21	SEQ ID NO：9439	0.00％	0.5
					3	53	SEQ ID NO：9440	0.00％	0.5

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	16	SEQ ID NO：9441	0.04％	2.5
2	71	SEQ ID NO：9442	0.04％	2.5
					3	47	SEQ ID NO：9443	0.02％	1.5
4	62	SEQ ID NO：9444	0.01％	0.9

5	20	SEQ ID NO：9445	0.00％	0.5
					6	38	SEQ ID NO：9446	0.00％	0.5

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	54	SEQ ID NO：9447	0.02％	2.7
2	17	SEQ ID NO：9448	0.01％	2
					3	3	SEQ ID NO：9449	0.01％	1.8

HLA A3-10mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	22	SEQ ID NO：9450	0.09％	12
2	16	SEQ ID NO：9451	0.01％	2
					3	54	SEQ ID NO：9452	0.00％	0.9

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	70	SEQ ID NO：9453	2.10％	33.6
2	7	SEQ ID NO：9454	1.12％	18
					3	60	SEQ ID NO：9455	0.46％	7.5
4	54	SEQ ID NO：9456	0.37％	6
					5	14	SEQ ID NO：9457	0.31％	5
6	19	SEQ ID NO：9458	0.30％	4.8
					7	47	SEQ ID NO：9459	0.30％	4.8
8	12	SEQ ID NO：9460	0.25％	4
					9	15	SEQ ID NO：9461	0.25％	4
10	67	SEQ ID NO：9462	0.25％	4
					11	21	SEQ ID NO：9463	0.18％	3
12	37	SEQ ID NO：9464	0.06％	1
					13	27	SEQ ID NO：9465	0.03％	0.5

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	14	SEQ ID NO：9466	12.52％	200
2	7	SEQ ID NO：9467	0.93％	15
					3	11	SEQ ID NO：9468	0.75％	12
4	60	SEQ ID NO：9469	0.56％	9
					5	18	SEQ ID NO：9470	0.45％	7.2

6	46	SEQ ID NO：9471	0.45％	7.2
					7	53	SEQ ID NO：9472	0.37％	6
8	69	SEQ ID NO：9473	0.35％	5.6
					9	66	SEQ ID NO：9474	0.25％	4
10	20	SEQ ID NO：9475	0.12％	2
					11	47	SEQ ID NO：9476	0.07％	1.2
12	36	SEQ ID NO：9477	0.06％	1
					13	26	SEQ ID NO：9478	0.04％	0.75
14	70	SEQ ID NO：9479	0.04％	0.72

HLA A 0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	54	SEQ ID NO：9480	0.02％	881.199
2	26	SEQ ID NO：9481	0.00％	95.013
					3	61	SEQ ID NO：9482	0.00％	93.69648
4	19	SEQ ID NO：9483	0.00％	40.2894864
					5	74	SEQ ID NO：9484	0.00％	12.6684
6	35	SEQ ID NO：9485	0.00％	10.34586
					7	69	SEQ ID NO：9486	0.00％	3.3704706
8	13	SEQ ID NO：9487	0.00％	1.656
					9	15	SEQ ID NO：9488	0.00％	1.47537042
10	68	SEQ IDNO：9489	0.00％	0.966
					11	22	SEQ ID NO：9490	0.00％	0.942678
12	12	SEQ ID NO：9491	0.00％	0.7669695
					13	36	SEQ ID NO：9492	0.00％	0.52661835

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	61	SEQ ID NO：9493	0.00％	93.69648
2	25	SEQ ID NO：9494	0.00％	63.33035625
					3	34	SEQ ID NO：9495	0.00％	50.232
4	53	SEQ ID NO：9496	0.00％	45.2838375
					5	26	SEQ ID NO：9497	0.00％	14.35752
6	27	SEQ ID NO：9498	0.00％	2.8557858
					7	17	SEQ ID NO：9499	0.00％	2.3973222
8	36	SEQ ID NO：9500	0.00％	1.798209
					9	69	SEQ ID NO：9501	0.00％	1.03521597
10	67	SEQ ID NO：9502	0.00％	0.966
					11	68	SEQ ID NO：9503	0.00％	0.910938
12	11	SEQ ID NO：9504	0.00％	0.7669695

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	17	SEQ ID NO：9505	2.22％	0.8

HLA A 1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	16	SEQ ID NO：9506	5.55％	2

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	27	SEQ ID NO：9507	0.37％	20
2	54	SEQ ID NO：9508	0.22％	12
					3	70	SEQ ID NO：9509	0.22％	12
4	67	SEQ ID NO：9510	0.11％	6
					5	12	SEQ ID NO：9511	0.07％	4
6	15	SEQ ID NO：9512	0.07％	4
					7	19	SEQ ID NO：9513	0.07％	4
8	49	SEQ ID NO：9514	0.03％	2
					9	69	SEQ ID NO：9515	0.03％	1.8
10	47	SEQ ID NO：9516	0.02％	1.2
					11	5	SEQ ID NO：9517	0.01％	1
12	9	SEQ ID NO：9518	0.01％	1
					13	35	SEQ ID NO：9519	0.01％	1
14	37	SEQ ID NO：9520	0.01％	0.6
					15	68	SEQ ID NO：9521	0.01％	0.6

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	69	SEQ ID NO：9522	0.66％	36
2	53	SEQ ID NO：9523	0.22％	12
					3	5	SEQ ID NO：9524	0.13％	7.5
4	66	SEQ ID NO：9525	0.11％	6
					5	11	SEQ ID NO：9526	0.07％	4
6	27	SEQ ID NO：9527	0.07％	4
					7	46	SEQ ID NO：9528	0.07％	4
8	18	SEQ ID NO：9529	0.02％	1.2
					9	9	SEQ ID NO：9530	0.01％	1
10	26	SEQ ID NO：9531	0.01％	1

11	25	SEQ ID NO：9532	0.01％	0.75
					12	17	SEQ ID NO：9533	0.01％	0.6
13	36	SEQ ID NO：9534	0.01％	0.6
					14	68	SEQ ID NO：9535	0.01％	0.6
15	35	SEQ ID NO：9536	0.00％	0.5
					16	42	SEQ ID NO：9537	0.00％	0.5
17	73	SEQ ID NO：9538	0.00％	0.5

The epi-position of table 25:SEQ ID NO:6052

HLA A1-9mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	365	SEQ ID NO：9539	0.8％	45
2	397	SEQ ID NO：9540	0.44％	25
					3	229	SEQ ID NO：9541	0.32％	18
4	103	SEQ ID NO：9542	0.17％	10
					5	338	SEQ ID NO：9543	0.17％	10
6	251	SEQ ID NO：9544	0.16％	9
					7	79	SEQ ID NO：9545	0.11％	6.25
8	119	SEQ ID NO：9546	0.10％	6
					9	361	SEQ ID NO：9547	0.08％	5
10	60	SEQ ID NO：9548	0.04％	2.25
					11	101	SEQ ID NO：9549	0.04％	2.25
12	278	SEQ ID NO：9550	0.04％	2.25
					13	23	SEQ ID NO：9551	0.02％	1.25
14	164	SEQ ID NO：9552	0.02％	1.25
					15	165	SEQ ID NO：9553	0.02％	1.25
16	295	SEQ ID NO：9554	0.02％	1.25
					17	172	SEQ ID NO：9555	0.01％	0.9
18	0	SEQ ID NO：9556	0.01％	0.75
					19	311	SEQ ID NO：9557	0.01％	0.75
20	78	SEQ ID NO：9558	0.01％	0.625

HLA A1-10mers
					Use the maximum possible of this molecule type to score				5625
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	114	SEQ ID NO：9559	1.11％	62.5
2	134	SEQ ID NO：9560	0.8％	45
					3	365	SEQ ID NO：9561	0.8％	45
4	77	SEQ ID NO：9562	0.66％	37.5
					5	103	SEQ ID NO：9563	0.44％	25

6	23	SEQ ID NO：9564	0.22％	12.5
					7	338	SEQ ID NO：9565	0.17％	10
8	361	SEQ ID NO：9566	0.17％	10
					9	324	SEQ ID NO：9567	0.11％	6.25
10	375	SEQ ID NO：9568	0.11％	6.25
					11	79	SEQ ID NO：9569	0.04％	2.5
12	295	SEQ ID NO：9570	0.04％	2.5
					13	346	SEQ ID NO：9571	0.04％	2.5
14	378	SEQ ID NO：9572	0.03％	2
					15	251	SEQ ID NO：9573	0.03％	1.8
16	214	SEQ ID NO：9574	0.02％	1.125
					17	160	SEQ ID NO：9575	0.01％	1
18	172	SEQ ID NO：9576	0.01％	0.9
					19	229	SEQ ID NO：9577	0.01％	0.9
20	376	SEQ ID NO：9578	0.01％	0.9

HLA A3-9mers
					Use the maximum possible of this molecule type to score				12150
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	229	SEQ ID NO：9579	0.49％	60
2	361	SEQ ID NO：9580	0.27％	33.75
					3	330	SEQ ID NO：9581	0.16％	20
4	218	SEQ ID NO：9582	0.09％	12
					5	338	SEQ ID NO：9583	0.04％	6
6	352	SEQ ID NO：9584	0.04％	6
					7	103	SEQ ID NO：9585	0.04％	5.4
8	291	SEQ ID NO：9586	0.01％	2
					9	241	SEQ ID NO：9587	0.01％	1.8
10	290	SEQ ID NO：9588	0.01％	1.8
					11	316	SEQ ID NO：9589	0.01％	1.8
12	222	SEQ ID NO：9590	0.01％	1.35
					13	266	SEQ ID NO：9591	0.01％	1.35
14	53	SEQ ID NO：9592	0.00％	1
					15	100	SEQ ID NO：9593	0.00％	0.9
16	138	SEQ ID NO：9594	0.00％	0.9
					17	240	SEQ ID NO：9595	0.00％	0.9
18	119	SEQ ID NO：9596	0.00％	0.675
					19	44	SEQ ID NO：9597	0.00％	0.6
20	161	SEQ IDNO：9598	0.00％	0.6

HLA A3-10mers
		Use the maximum possible of this molecule type to score	12150

Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	338	SEQ ID NO：9599	0.49％	60
2	160	SEQ ID NO：9600	0.32％	40
					3	352	SEQ ID NO：9601	0.24％	30
4	361	SEQ ID NO：9602	0.18％	22.5
					5	103	SEQ ID NO：9603	0.13％	16.2
6	290	SEQ ID NO：9604	0.07％	9
					7	351	SEQ ID NO：9605	0.07％	9
8	44	SEQ ID NO：9606	0.04％	6
					9	228	SEQ ID NO：9607	0.03％	4.05
10	394	SEQ ID NO：9608	0.02％	3
					11	240	SEQ ID NO：9609	0.02％	2.7
12	100	SEQ ID NO：9610	0.01％	1.8
					13	114	SEQ ID NO：9611	0.01％	1.8
14	93	SEQ ID NO：9612	0.01％	1.5
					15	134	SEQ ID NO：9613	0.01％	1.5
16	221	SEQ ID NO：9614	0.01％	1.35
					17	330	SEQ ID NO：9615	0.00％	1.2
18	112	SEQ ID NO：9616	0.00％	0.9
					19	218	SEQ ID NO：9617	0.00％	0.9
20	55	SEQID NO：9618	0.00％	0.6

HLA A24-9mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	345	SEQ ID NO：9619	1.50％	24
2	306	SEQ ID NO：9620	0.75％	12
					3	222	SEQ ID NO：9621	0.54％	8.64
4	111	SEQ ID NO：9622	0.51％	8.25
					5	159	SEQ ID NO：9623	0.45％	7.2
6	219	SEQ ID NO：9624	0.45％	7.2
					7	283	SEQ ID NO：9625	0.45％	7.2
8	266	SEQ ID NO：9626	0.42％	6.72
					9	56	SEQ ID NO：9627	0.41％	6.6
10	131	SEQ ID NO：9628	0.37％	6
					11	214	SEQ ID NO：9629	0.37％	6
12	297	SEQ ID NO：9630	0.37％	6
					13	86	SEQ ID NO：9631	0.31％	5
14	122	SEQ ID NO：9632	0.31％	5
					15	48	SEQ ID NO：9633	0.30％	4.8
16	105	SEQ ID NO：9634	0.30％	4.8
					17	213	SEQ ID NO：9635	0.30％	4.8

18	323	SEQ ID NO：9636	0.30％	4.8
					19	338	SEQ ID NO：9637	0.30％	4.8
20	399	SEQ ID NO：9638	0.30％	4.8

HLA A24-10mers
					Use the maximum possible of this molecule type to score				1596.672
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	65	SEQ ID NO：9639	0.93％	15
2	306	SEQ ID NO：9640	0.75％	12
					3	95	SEQ ID NO：9641	0.66％	10.56
4	36	SEQ ID NO：9642	0.60％	9.6
					5	385	SEQ ID NO：9643	0.50％	8
6	111	SEQ ID NO：9644	0.46％	7.5
					7	104	SEQ ID NO：9645	0.45％	7.2
8	214	SEQ ID NO：9646	0.45％	7.2
					9	221	SEQ ID NO：9647	0.45％	7.2
10	277	SEQ ID NO：9648	0.45％	7.2
					11	150	SEQ ID NO：9649	0.37％	6
12	152	SEQ ID NO：9650	0.37％	6
					13	158	SEQ ID NO：9651	0.37％	6
14	171	SEQ ID NO：9652	0.37％	6
					15	343	SEQ ID NO：9653	0.37％	6
16	110	SEQ ID NO：9654	0.34％	5.5
					17	85	SEQ ID NO：9655	0.31％	5
18	47	SEQ ID NO：9656	0.30％	4.8
					19	213	SEQ ID NO：9657	0.30％	4.8
20	218	SEQ ID NO：9658	0.30％	4.8

HLA A 0201-9mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	222	SEQ ID NO：9659	0.03％	1267.10434728
2	226	SEQ ID NO：9660	0.00％	69.552
					3	316	SEQ ID NO：9661	0.00％	50.232
4	351	SEQ ID NO：9662	0.00％	31.24872
					5	159	SEQ ID NO：9663	0.00％	13.6235739
6	406	SEQ ID NO：9664	0.00％	11.4264
					7	165	SEQ ID NO：9665	O.00％	8.14407
8	238	SEQ ID NO：9666	0.00％	7.0518
					9	138	SEQ ID NO：9667	0.00％	5.112072
10	130	SEQ ID NO：9668	0.00％	3.00547233
					11	303	SEQ ID NO：9669	0.00％	2.59578

12	157	SEQ ID NO：9670	0.00％	2.412585
					13	219	SEQ ID NO：9671	0.00％	2.103255861
14	305	SEQ ID NO：9672	0.00％	1.86369
					15	158	SEQ ID NO：9673	0.00％	1.646892
16	331	SEQ ID NO：9674	0.00％	1.614048
					17	399	SEQ ID NO：9675	0.00％	1.442246832
18	324	SEQ ID NO：9676	0.00％	1.319625
					19	312	SEQ ID NO：9677	0.00％	1.233099
20	262	SEQ ID NO：9678	0.00％	0.966

HLA A 0201-10mers
					Use the maximum possible of this molecule type to score				3925227.1
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	221	SEQ ID NO：9679	0.00％	309.0498408
2	112	SEQ ID NO：9680	0.00％	98.26704
					3	330	SEQ ID NO：9681	0.00％	98.26704
4	158	SEQ ID NO：9682	0.00％	36.31608
					5	218	SEQ ID NO：9683	0.00％	24.0754248
6	124	SEQ ID NO：9684	0.00％	12.2199
					7	55	SEQ ID NO：9685	0.00％	10.467576
8	315	SEQ ID NO：9686	0.00％	7.7274204
					9	350	SEQ ID NO：9687	0.00％	4.296699
10	405	SEQ ID NO：9688	0.00％	4.286487
					11	388	SEQ ID NO：9689	0.00％	4.054785
12	322	SEQ ID NO：9690	0.00％	3.883803
					13	130	SEQ ID NO：9691	0.00％	3.428691903
14	45	SEQ ID NO：9692	0.00％	3.411230625
					15	132	SEQ ID NO：9693	0.00％	2.99943
16	410	SEQ ID NO：9694	0.00％	2.63718
					17	316	SEQ ID NO：9695	0.00％	2.48686074
18	104	SEQ ID NO：9696	0.00％	2.477311485
					19	164	SEQ ID NO：9697	0.00％	2.2011
20	282	SEQ ID NO：9698	0.00％	2.16591

HLA A 1101-9mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	361	SEQ ID NO：9699	16.66％	6
2	53	SEQ ID NO：9700	2.77％	1
					3	240	SEQ ID NO：9701	1.66％	0.6
4	241	SEQ ID NO：9702	1.66％	0.6

HLA A 1101-10mers
					Use the maximum possible of this molecule type to score				36
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	361	SEQ ID NO：9703	16.66％	6
2	93	SEQ ID NO：9704	8.33％	3
					3	338	SEQ ID NO：9705	3.33％	1.2
4	134	SEQ ID NO：9706	2.77％	1
					5	228	SEQ ID NO：9707	2.5％	0.9
6	160	SEQ ID NO：9708	2.22％	0.8
					7	239	SEQ ID NO：9709	1.66％	0.6
8	240	SEQ ID NO：9710	1.66％	0.6
					9	257	SEQ ID NO：9711	1.66％	0.6
10	379	SEQ ID NO：9712	1.66％	0.6

HLA B7-9mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	105	SEQ ID NO：9713	14.81％	800
2	66	SEQ ID NO：9714	1.48％	80
					3	93	SEQ ID NO：9715	0.92％	50
4	257	SEQ ID NO：9716	0.55％	30
					5	323	SEQ ID NO：9717	0.37％	20
6	211	SEQ ID NO：9718	0.22％	12
					7	219	SEQ ID NO：9719	0.22％	12
8	403	SEQ ID NO：9720	0.18％	10
					9	343	SEQ ID NO：9721	0.14％	8
10	12	SEQ ID NO：9722	0.11％	6
					11	113	SEQ ID NO：9723	0.11％	6
12	48	SEQ ID NO：9724	0.07％	4
					13	56	SEQ ID NO：9725	0.07％	4
14	150	SEQ ID NO：9726	0.07％	4
					15	153	SEQ ID NO：9727	0.07％	4
16	159	SEQ ID NO：9728	0.07％	4
					17	213	SEQ ID NO：9729	0.07％	4
18	216	SEQ ID NO：9730	0.07％	4
					19	222	SEQ ID NO：9731	0.07％	4
20	283	SEQ ID NO：9732	0.07％	4

HLA B7-10mers
					Use the maximum possible of this molecule type to score				5400
Ordering	Initiation site	Sequence	Account for the percentage that maximum is scored	Score
					1	36	SEQ ID NO：9733	1.48％	80

2	150	SEQ ID NO：9734	1.48％	80
					3	343	SEQ ID NO：9735	1.48％	80
4	12	SEQ ID NO：9736	1.11％	60
					5	308	SEQ ID NO：9737	1.11％	60
6	130	SEQ ID NO：9738	0.37％	20
					7	55	SEQ ID NO：9739	0.22％	12
8	210	SEQ ID NO：9740	0.22％	12
					9	218	SEQ ID NO：9741	0.22％	12
10	201	SEQ ID NO：9742	0.18％	10
					11	121	SEQ ID NO：9743	0.14％	8
12	391	SEQ ID NO：9744	0.13％	7.5
					13	112	SEQ ID NO：9745	0.11％	6
14	385	SEQ ID NO：9746	0.11％	6
					15	47	SEQ ID NO：9747	0.07％	4
16	66	SEQ ID NO：9748	0.07％	4
					17	95	SEQ ID NO：9749	0.07％	4
18	104	SEQ ID NO：9750	0.07％	4
					19	152	SEQ ID NO：9751	0.07％	4
20	158	SEQ ID NO：9752	0.07％	4

Table 26: the cloned sequence that is used for Bacillus coli expression

ORF	DNA length bp	The clone
				pET	pGEX
		P28	537			NdeI/XhoI
P65	1917			NheI/HindIII
		Nsp1A	2495			NheI/XhoI
Nsp1B	2153			NdeI/XhoI
		Nsp1C	2612			NdeI/XhoI
Nsp2A
		431	NdeI/XhoI	BamHI/XhoI
Nsp2B
		426	NdeI/XhoI	BamHI/XhoI
Nsp3					870	NdeI/XhoI
	Nsp4	249	NdeI/XhoI	BamHI/XhoI
Nsp5					594	NheI/XhoI
	Nsp6	339	NdeI/XhoI	BamHI/XhoI
Nsp7					417	NdeI/XhoI	BamHI/XhoI
	Nsp9A	1385	NheI/XhoI
Nsp9B					1409	NdeI/XhoI
	Nsp10	1803	NheI/XhoI
Nsp11					1581	NdeI/XhoI
	Nsp12	1038	NdeI/HindIII
Nsp13					897	NdeI/XhoI
	Furcella (S1)	1946	NdeI/XhoI
Furcella (S2)					1598	NdeI/XhoI
	Furcella (S1-S2)	3545	NdeI/XhoI
HR1					287	NdeI/XhoI	BamHI/XhoI
	HR2	146	NdeI/XhoI	BamHI/XhoI
ORF3 100					525	NdeI/XhoI
	ORF4	465	NdeI/XhoI
Coating (E)					231	NdeI/XhoI	BamHI/XhoI
	Matrix (M) 100	366	NdeI/XhoI	BamHI/XhoI
ORF7 18					137	NdeI/XhoI	BamHI/XhoI
	ORF8	369	NdeI/XhoI	BamHI/XhoI
ORF9					135	NdeIzXhoI	BamHI/XhoI
	ORF10	120	NheI/XhoI	BamHI/XhoI
ORF11					255	NdeI/XhoI	BamHI/XhoI
	Nucleocapsid (N)	1269	NdeI/EcoRI
ORF12					297	NdeI/EcoRI	BamHI/EcoRI

Table 27: primer

ORF	Forward primer	Reverse primer
			P28	9803	9818
P65	9804	9819
			Nsp1A	9805	9820
Nsp1B	9806	9821
			Nsp1C	9807	9822
Nsp2+Nsp3	9808	9823
			Nsp4-Nsp7	9809	9824
Nsp9A	9810	9825
			Nsp9B	9811	9826
Nsp10	9812	9827
			Nsp11	9813	9828
Nsp12-Nsp13	9814	9829
			ORF3-ORF4	9815	9830
Env-ORF10	9816	9831
			ORF11-ORF12	9817	9832

Table 28: primer

ORF	Forward primer	Reverse primer
			Nsp2A	SEQ ID NO：9833	SEQ ID NO：9858
Nsp2B	SEQ ID NO：9834	SEQ ID NO：9859
			Nsp3	SEQ ID NO：9835	SEQ ID NO：9860
Nsp4	SEQ ID NO：9836	SEQ ID NO：9861
			Nsp5	SEQ ID NO：9837	SEQ ID NO：9862
Nsp6	SEQ ID NO：9838	SEQ ID NO：9863
			Nsp7	SEQ ID NO：9839	SEQ ID NO：9864
Nsp12	SEQ ID NO：9840	SEQ ID NO：9865
			Nsp13	SEQ ID NO：9841	SEQ ID NO：9866
Furcella S1	SEQ ID NO：9842	SEQ ID NO：9867
			Furcella S2	SEQ ID NO：9843	SEQ ID NO：9868
Furcella S1-S2	SEQ ID NO：9844	SEQ ID NO：9869
			HR1	SEQ ID NO：9845	SEQ ID NO：9870
HR2	SEQ ID NO：9846	SEQ ID NO：9871
			Orf3Δ100	SEQ ID NO：9847	SEQ ID NO：9872
Orf4	SEQ ID NO：9848	SEQ ID NO：9873
			Env E	SEQ ID NO：9849	SEQ ID NO：9874
Matrix M Δ 100	SEQ ID NO：9850	SEQ ID NO：9875
			Orf7Δ18	SEQ ID NO：9851	SEQ ID NO：9876
Orf8	SEQ ID NO：9852	SEQ ID NO：9877
			Orf9	SEQ ID NO：9853	SEQ ID NO：9878
Orf10	SEQ ID NO：9854	SEQ ID NO：9879
			Orf11	SEQ ID NO：9855	SEQ ID NO：9880
Nucleocapsid N	SEQ ID NO：9856	SEQ ID NO：9881
			Orf12	SEQ ID NO：9857	SEQ ID NO：9882

Table 29: clone, purifying and at expression in escherichia coli

SARS CoV ORFs	M.W   Kd	The clone	Express	Purifying
					P28	19,7	+	+	his  sol
P65
		70,3	+	+	his sol
	Nsp1A (N-is terminal)					91,6	+	+	his ins
Nsp1B (core)		80,8	+	-
	Nsp1C (C-is terminal)					95,3	+	-
Nsp2A (N-is terminal)		15,8	+	+	his ins
	Nsp2B (C-is terminal)					15,5	+	+	his  sol
Nsp3
		31,9	+	-
	Nsp4					9,1	+	+	his  sol
Nsp5
		21,8	+	+	his  sol
	Nsp6
		12,4	+	+	his  sol
Nsp7
		15,3	+	+	his ins
	Nsp9A (N-is terminal)					50,8	+	-
Nsp9B (C-is terminal)		51,6	+	+	his ins
	Nsp10					66	-
Nsp11		58	-
	Nsp12					38	-
Nsp13		32,7	+	+	his ins
	Furcella (S1-his)					71,3	+	+	his ins
Furcella (S2-his)		58,6	The clone	-
	Furcella (S1S2-his)					130	+	+	his ins
HR1		11	+	+	his ins
	HR2					5,4	+	+	his  sol
ORF3Δ100
1		19,1	+	-
	ORF4					16,9	+	+	His ins (trisome)
Coating (E)		34,3	+	+	gst ins(IB)
	Matrix (M) Δ 100					13,3	+	+	his ins
ORF7Δ18 2		31	+	+	gst sol
	ORF8					39,5	+	+	gst ins(IB)
ORF9		30,8	+	+	gst sol
	ORF10					30,3	+	+	gst ins(IB)
ORF11		35,2	+	+	gst ins(IB)
	Nucleocapsid (N)					43,6	+	+	his ins
ORF12		36,7	+	+	his ins

Table 30: Bacillus coli expression, purifying and output

Protein	Mark	Purity (%)	Output (mg/l)
				Nsp2A (N-is terminal)	His	95	1.7
Nsp2B (C-is terminal)	His	95	4.1
				Nsp4	His	95	12.6
Nsp5	His	95	5.88
				Nsp6	His	95	8.1
P28	His	95	1
				P65	His	80	0.553
HR2	His	95	11.9
				HR1	His	80	2.64
Nsp1A	His	95	0.267
				Furcella S1-S2	His	80	0.381
Matrix M	His	85	12.4
				ORF7	GST	85	4.9

Table 31: primer

SEQ ID NO：	Ordering	Model	Local	(position)
					10235	F1	1	1	(106)
10236	F2	2	1	(728)
					10237	F3	3	1	(112)
10238	F4	5	2	(1331)
					10239	F5	6	1	(12)
10240	F6	6	1	(346)
					10241	F7	8	1	(904)
10242	F8	9	1	(1016)
					10243	F9	9	1	(1015)
10244	F10	9	1	(719)
					10245	F11	9	1	(720)
10246	F12	10	1	(724)
					10247	R1	2	1	(1283)
10248	R2	4	1	(756)
					10249	R3	4	1	(758)
10250	R4	5	2	(259)
					10251	R5	6	1	(54)
10252	R6	7	1	(648)
					10253	R7	8	1	(948)
10254	R8	8	1	(260)
					10255	R9	9	1	(1282)
10256	R10	9	1	(950)
					10257	R11	9	1	(756)
10258	R12	10	1	(132)

Table 32: primer

Primer tabulation: (forward)
						Ordering	Score		Sequence	(position)
Model	Local
		F1   F2   F3   F4   F5   F6   F7   F8   F9   F10   F11   F12   F13   F14   F15   F16   F17   F18   F19   F20   F21   F22   F23   F24   F25	7   7   7   7   7   9   9   10   11   11   12   12   14   14   16   17   17   17   17   20   20   28   28   29   29	1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1	SEQ ID NO：10352   SEQ ID NO：10353   SEQ ID NO：10354   SEQ ID NO：10355   SEQ ID NO：10356   SEQ ID NO：10357   SEQ ID NO：10358   SEQ ID NO：10359   SEQ ID NO：10360   SEQ ID NO：10361   SEQ ID NO：10362   SEQ ID NO：10363   SEQ ID NO：10364   SEQ ID NO：10365   SEQ ID NO：10366   SEQ ID NO：10367   SEQ ID NO：10368   SEQ ID NO：10369   SEQ ID NO：10370   SEQ ID NO：10371   SEQ ID NO：10372   SEQ ID NO：10373   SEQ ID NO：10374   SEQ ID NO：10375   SEQ ID NO：10376		(290)   (291)   (294)   (292)   (293)   (198)   (199)   (33)   (200)   (299)   (298)   (297)   (35)   (34)   (300)   (295)   (296)   (175)   (36)   (202)   (201)   (204)   (203)   (269)   (268)
Primer tabulation (oppositely)
						Ordering	Model	Local	Sequence	(position)
R1   R2   R3   R4   R5   R6   R7   R8   R9   R10   R11   R12   R13   R14   R15   R16   R17   R18   R19   R20   R21   R22   R23   R24   R25	7   9   11   11   12   12   13   14   14   15   16   17   17   17   17   18   20   20   21   22   28   29   32   35   36	1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1	SEQ ID NO：10377   SEQ ID NO：10378   SEQ ID NO：10379   SEQ ID NO：10380   SEQ ID NO：10381   SEQ ID NO：10382   SEQ ID NO：10383   SEQ ID NO：10384   SEQ ID NO：10385   SEQ ID NO：10386   SEQ ID NO：10387   SEQ ID NO：10388   SEQ ID NO：10389   SEQ ID NO：10390   SEQ ID NO：10391   SEQ ID NO：10392   SEQ ID NO：10393   SEQ ID NO：10394   SEQ ID NO：10395   SEQ ID NO：10396   SEQ ID NO：10397   SEQ ID NO：10398   SEQ ID NO：10399   SEQ ID NO：10400   SEQ ID NO：10401	(337)   (229)   (230)   (338)   (207)   (338)   (231)   (80)   (232)   (82)   (340)   (83)   (206)   (82)   (337)   (341)   (340)   (233)   (79)   (213)   (236)   (317)   (391)   (57)   (237)
						Primer tabulation (left side): SEQ ID NOs：10402-10433					Primer tabulation (right side): SEQ ID NOs：10434-10464
Primer tabulation (forward): SEQ ID NOs：10465-10484					Primer tabulation (oppositely): SEQ ID NOs：10485-10504

Table 33: primer

Primer tabulation (forward)
					Ordering	Score		Sequence	(position)
Model	Local
		F1   F2   F3   F4   F5   F6   F7   F8   F9   F10   F11   F12   F13   F14   F15   F16   F17   F18   F19   F20   F21   F22   F23   F24   F25   F26   F27   F28   F29   F30   F31   F32   F33   F34   F35   F36   F37   F38   F39   F40   F41   F42   F43   F44   F45   F46   F47   F48   F49   F50	1   2   2   3   4   4   4   5   5   5   5   6   6   6   6   6   6   6   6   7   7   7   7   7   7   7   7   7   8   8   8   8   8   8   8   9   9   9   9   9   9   10   10   10   10   10   10   11   11   11	1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1		SEQ ID NO：10580   SEQ ID NO：10581   SEQ ID NO：10582   SEQ ID NO：10583   SEQ ID NO：10584   SEQ ID NO：10585   SEQ ID NO：10586   SEQ ID NO：10587   SEQ ID NO：10588   SEQ ID NO：10589   SEQ ID NO：10590   SEQ ID NO：10591   SEQ ID NO：10592   SEQ ID NO：10593   SEQ ID NO：10594   SEQ ID NO：10595   SEQ ID NO：10596   SEQ ID NO：10597   SEQ ID NO：10598   SEQ ID NO：10599   SEQ ID NO：10600   SEQ ID NO：10601   SEQ ID NO：10602   SEQ ID NO：10603   SEQ ID NO：10604   SEQ ID NO：10605   SEQ ID NO：10606   SEQ ID NO：10607   SEQ ID NO：10608   SEQ ID NO：10609   SEQ ID NO：10610   SEQ ID NO：10611   SEQ ID NO：10612   SEQ ID NO：10613   SEQ ID NO：10614   SEQ ID NO：10615   sEQ ID NO：10616   SEQ ID NO：10617   SEQ ID NO：10618   SEQ ID NO：10619   SEQ ID NO：10620   SEQ ID NO：10621   SEQ ID NO：10622   SEQ ID NO：10623   SEQ ID NO：10624   SEQ ID NO：10625   SEQ ID NO：10626   SEQ ID NO：10627   SEQ ID NO：10628   SEQ ID NO：10629	(637)   (439)   (440)   (729)   (696)   (697)   (111)   (867)   (868)   (869)   (640)   (438)   (437)   (436)   (732)   (635)   (457)   (458)   (636)   (854)   (855)   (581)   (853)   (342)   (343)   (112)   (94)   (642)   (638)   (639)   (730)   (641)   (731)   (326)   (325)   (517)   (701)   (208)   (209)   (702)   (210)   (634)   (694)   (693)   (728)   (695)   (95)   (455)   (456)   (454)

Primer tabulation (oppositely)
						Ordering	Score		Sequence	(position)
Model	Local
		R1   R2   R3   R4   R5   R6   R7   R8   R9   R10   R11   R12   R13   R14   R15   R16   R17   R18   R19   R20   R21   R22   R23   R24   R25   R26   R27   R28   R29   R30   R31   R32   R33   R34   R35   R36   R37   R38   R39   R40   R41   R42   R43   R44   R45   R46   R47   R48   R49   R50	1   1   2   3   3   4   4   4   4   4   5   5   6   6   6   6   6   6   7   7   7   7   7   7   7   7   8   8   8   8   8   9   9   9   9   10   10   10   10   10   11   11   11   11   11   11   12   12   12   12	1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1   1	SEQ ID NO：10630   SEQ ID NO：10631   SEQ ID NO：10632   SEQ ID NO：10633   SEQ ID NO：10634   SEQ ID NO：10635   SEQ ID NO：10636   SEQ ID NO：10637   SEQ ID NO：10638   SEQ ID NO：10639   SEQ ID NO：10640   SEQ ID NO：10641   SEQ ID NO：10642   SEQ ID NO：10643   SEQ ID NO：10644   SEQ ID NO：10645   SEQ ID NO：10646   SEQ ID NO：10647   SEQ ID NO：10648   SEQ ID NO：10649   SEQ ID NO：10650   SEQ ID NO：10651   SEQ ID NO：10652   SEQ ID NO：10653   SEQ ID NO：10654   SEQ ID NO：10655   SEQ ID NO：10656   SEQ ID NO：10657   SEQ ID NO：10658   SEQ ID NO：10659   SEQ ID NO：10660   SEQ ID NO：10661   SEQ ID NO：10662   SEQ ID NO：10663   SEQ ID NO：10664   SEQ ID NO：10665   SEQ ID NO：10666   SEQ ID NO：10667   SEQ ID NO：10668   SEQ ID NO：10669   SEQ ID NO：10670   SEQ ID NO：10671   SEQ ID NO：10672   SEQ ID NO：10673   SEQ ID NO：10674   SEQ ID NO：10675   SEQ ID NO：10676   SEQ ID NO：10677   SEQ ID NO：10678   SEQ ID NO：10679		(367)   (666)   (464)   (669)   (750)   (720)   (465)   (370)   (668)   (135)   (901)   (667)   (609)   (464)   (665)   (486)   (356)   (758)   (366)   (368)   (136)   (675)   (366)   (608)   (884)   (120)   (355)   (671)   (756)   (751)   (666)   (242)   (543)   (724)   (482)   (121)   (662)   (750)   (719)   (242)   (484)   (375)   (728)   (373)   (998)   (486)   (881)   (882)   (244)   (1003)
Primer tabulation (left side): SEQ ID NOs：10680-10974						Primer tabulation (right side): SEQ ID NOs：10975-11282
					Primer tabulation (forward): SEQ ID NOs：11283-11302					Primer tabulation (oppositely): SEQ ID NOs：11303-11322

Table 34

Table 35

Table 35 is continuous

Table 35 is continuous

Table 35 is continuous

The sequence summary

SEQ ID NO：	Describe
		1	TOR from British Columbia Province，Canada genome scientific center2The genome assembling sketch of separator sequence. TOR2_draft_genome_assembly_120403 Release I
2	CDC SARS-CoV strain sequence. complete nucleotide sequence (Urbani strain)
		3-20	Group-specific coronavirus gene outcome＞feline infectious peritonitis virus (FIPV) 3/4=ORF 3b; 5/6=ORF 3X; 7/8=ORF 3A＞canine coronavirus 9/10=ORF 7b; 11/12=ORF 7a＞avian infectious bronchitis virus 13/14=ORF 5b; 15/16=ORF 5a; 17/18=ORF 3a; 19/20=ORF 3b
21-520	500 primers of left-hand component
		521-1020	500 primers of right-hand component
1021-3520	The forward primer of table 4
		3521-6020	The reverse primer of table 4
6021-6026	The primer of Fig. 9
		6027-6033	The primer of Figure 11
6034-6038	Primer http://content.nejm.org/cgi/reprint/NEJMoa030781v2.pdf from following network address
		6039-6051	PEP1 to PEP13
6052	The PEP13 that prolongs
		6053-6056	229E human corona virus sequence
6057-6060	The TGV sequence
		6061-6064	The PEDV sequence
6065-6068	The bovine coronavirus sequence
		6069-6071	The murine hepatitis virus sequence
6072-6075	The AIBV sequence
		6076-6170	Primer sequence (forward)
6171-6265	Primer sequence (oppositely)
		6266-6304	Primer sequence (forward)
6305-6343	Primer sequence (oppositely)
		6344-6366	Primer sequence (forward)
6367-6392	Primer sequence (oppositely)
		6393-6440	Primer sequence (forward) F1-F48
6441-6487	Primer sequence (oppositely) R1-R47
		6488-6559	Primer sequence
6560-6568	Primer sequence
		6569	Nsp2 protease (3CL-PRO) sequence in the sars coronavirus
6570-72	The nsp2 protease (3CLp) of ox IBV, MHV and BcoV
		6573	Nsp2 protease consensus sequence
6574-6577	The IG sequence of Figure 18
		6578	NSh among the pCMVIII expresses construction
6579	NS among the pCMVIII expresses construction
		6580	NSh Δ TC among the pCMVIII expresses construction
6581	NS Δ TC among the pCMVIII expresses construction
		6582	NS1h among the pCMVIII expresses construction
6583	NS1 among the pCMVIII expresses construction
		6584-6585	The cDNA amplimer
6585-6587	The RT-PCR primer
		6588-6809	The composition sequence of Figure 23 (〉=4 amino acid)
6810-7179	The composition sequence of Figure 24 (〉=4 amino acid)
		7180-7187	N-glycosylation site in the SEQ ID NO:6039

7188-7189	The composition sequence of Figure 25
		7190	The fragment of SEQ ID NO:7188
7191	The polynucleotides of coding SEQ ID NO:7190
		7192	The amino acid 879-1005 of SEQ ID NO:6042
7193	The amino acid 879-980 of SEQ ID NO:6042
		7194	The amino acid 901-1005 of SEQ ID NO:6042
7195	The amino acid/11 144-1201 of SEQ ID NO:6042
		7196	The amino acid/11 144-1196 of SEQ ID NO:6042
7197-7199	Film fusogenic peptide zone
		7200-7206	Polypeptide based on NadA
7207-7223	N-glycosylation site in the SEQ ID NO:6042
		7224-7231	Sliding area
7232	The Orflab polyprotein
		7233-7244	The Orflab polyprotein
7245-7247	The X of SEQ ID NOS 7233-72442Sequence
		7248-7253	The Orflab polyprotein
7254	2 of zinc lands
		7255-7271	SEQ ID NOS:6040-41,6043,6045-46, the N-glycosylation site among the 6050-51
7272-7291	Polypeptide and polynucleotides
		7292-7293	Intergenic sequence
7294-7301	Nucleotides between SARSV genome 5 ' terminal gene after the sequence
		7302-7306	The NadA construction
7307-7308	The fragment of SEQ ID NO:6042
		7309	The NadA sequence
7310-7311	The NadA targeting sequencing
		7312-7315	The amino acid sequence of NadA
7316-7324	The PCR primer
		7325-7330	Primer
7331	The CCACC sequence
		7332-7336	3 ' UTR forward primer
7337-7341	3 ' UTR reverse primer
		7342-7352	3 ' UTR probe
7353-7362	5 ' UTR forward primer
		7363-7373	5 ' UTR reverse primer
7374-7385	5 ' UTR probe
		7386	8 conservative nucleotides
7387	The reverse component of SEQ ID NO:7293
		7388	Intergenic sequence
7389	Poly T
		7390	Stem-ring sequence
7391-7392	Many glycine joint
		7393	The polyhistidine mark
7394	Nucleocapsid epi-position site
		7395	Antisense primer
7396-7397	Probe
		7398-7399	The antigenicity fragment of SEQ ID NO:6042
7400-7639	The T epitope analysis of SEQ ID NO:6039
		7640-7800	The T epitope analysis of SEQ ID NO:6040
7801-8040	The T epitope analysis of SEQ ID NO:6041
		8041-8280	The T epitope analysis of SEQ ID NO:6042
8281-8486	The T epitope analysis of SEQ ID NO:6043
		8487-8665	The T epitope analysis of SEQ ID NO:6044
8666-8820	The T epitope analysis of SEQ ID NO:6045

8821-9018	The T epitope analysis of SEQ ID NO:6046
		9019-9131	The T epitope analysis of SEQ ID NO:6047
9132-9308	The T epitope analysis of SEQ ID NO:6048
		9309-9437	The T epitope analysis of SEQ ID NO:6049
9438-9538	The T epitope analysis of SEQ ID NO:6050
		9539-9752	The T epitope analysis of SEQ ID NO:6052
9753-9763	The spike protein amplimer is particularly useful for the furcella fragment
		9764-9765	N-glycosylation site among the SEQ ID NO:6039
9766-9779	ORFlab cleaved products (table 10)
		9780-9782	Forward primer, reverse primer, probe
9783-9784	Be rich in the zone of lysine
		9785-9798	Be used for the oligonucleotides that saccharomyces cerevisiae is expressed
9799-9802	Figure 65 and 66 sequence
		9803-9882	The primer that is used for escherichia coli cloning
9883-9885	Fig. 3 A, 3B, the BCV nucleotide sequences of 3C
		9886-9891	Fig. 4 A, 4B, 4C, 4D, 4E, the BCV amino acid sequence of 4F
9892	BCV 5′UTR
		9893	BCV 3′UTR
9894-9896	Fig. 3 A, 3B, the MHV nucleotide sequences of 3C
		9897-9902	Fig. 4 A, 4B, 4C, 4D, 4E, the MHV amino acid sequence of 4F
9903-9904	Fig. 3 A, the AIBV nucleotide sequences of 3B
		9905-9909	Fig. 4 A, 4B, 4D, 4E, the AIBV amino acid sequence of 4F
9910	AIBV 5′UTR
		9911	AIBV 3′UTR
9912-9913	Fig. 3 B, the HOBMPRO of 3C, HOBHEGA nucleotide sequences
		9914-9918	Fig. 4 A, 4B, 4C, 4E, the people CoV amino acid sequence of 4F
9919	HCoV-OC43 5′UTR
		9920	HCoV-OC43 3′UTR
9921-9923	The pCMVKm2 carrier
		9924-9926	The preferred N of codon, M and E sequence
9927	BNI-1
		9928-9959	From SEQ ID NO:9927 release 〉=the composition amino acid sequence of 4aa
9960	The ORFlab variant
		9961	The ORFla variant
9962	The spike protein variant
		9963	The memebrane protein variant
9964	The nucleocapsid protein variant
		9965-9966	End ORF
9967	FRA complete genome group

Claims (119)

1. the isolated polypeptide of a SARS virus.

2. polypeptide as claimed in claim 1, wherein, described polypeptide is furcella (S) polypeptide, coating (E) polypeptide, film (M) polypeptide, hemagglutinin-esterase polypeptide (HE), nucleocapsid (N) polypeptide, ORF1a polypeptide, ORF1ab polypeptide, the proteolytic fragments of ORF1a polypeptide or the proteolytic fragments of ORF1ab polypeptide.

3. polypeptide as claimed in claim 1, wherein, described polypeptide comprises and is selected from lower group amino acid sequence: SEQ ID NOs:6039,7232,9766,9767,9768,9769,9770,9771,9772,9773,9774,9775,9776,9777,9778,9779,6042,6043,6044,6045,6046,6047,6048,6049,6050 or 6052.

4. polypeptide as claimed in claim 1, wherein, described polypeptide comprises with the amino acid sequence that is selected from lower group to be had＞amino acid sequence of 75% sequence homogeny: SEQ ID NOS:6042,6043,6044,6045,6046,6047,6048,6049,6050,6052,9766,9767,9768,9769,9770,9771,9772,9773,977,4,9775,9776,9777,9778,9779,9997,9998,10149,10316,10338,10339,10340,10341,10342,10532,10533,10571,10572,10573,10574,10575,10576,10577,10578,10579,11561,11562,11618,11619,11620,11627,11630,11633 and 11636.

5. polypeptide as claimed in claim 1, wherein, described polypeptide comprises the fragment of at least 10 continuous amino acids of the amino acid sequence that is selected from lower group: SEQ ID NOS:6042,6043,6044,6045,6046,6047,6048,6049,6050,6052,9766,9767,9768,9769,9770,9771,9772,9773,9774,9775,9776,9777,9778,9779,9997,9998,10149,10316,10338,10339,10340,10341,10342,10532,10533,10571,10572,10573,10574,10575,10576,10577,10578,10579,11552,11561,11562,11618,11619,11620,11627,11630,11633 and 11636.

6. one kind comprises with the amino acid sequence that is selected from lower group and has＞polypeptide of the amino acid sequence of 80% sequence homogeny: SEQ ID NOS:6042,6043,6044,6045,6046,6047,6048,6049,6050,6052,9766,9767,9768,9769,9770,9771,9772,9773,9774,9775,9776,9777,9778,9779,9997,9998,10149,10316,10338,10339,10340,10341,10342,10532,10533,10571,10572,10573,10574,10575,10576,10577,10578,10579,11552,11561,11562,11618,11619,11620,11627,11630,11633 and 11636.

7. the polypeptide of the amino acid sequence of a fragment that comprises at least 10 continuous amino acids that contain the amino acid sequence that is selected from lower group: SEQ ID NOS:6042,6043,6044,6045,6046,6047,6048,6049,6050,6052,9766,9767,9768,9769,9770,9771,9772,9773,9774,9775,9776,9777,9778,9779,9997,9998,10149,10316,10338,10339,10340,10341,10342,10532,10533,10571,10572,10573,10574,10575,10576,10577,10578,10579,11552,11561,11562,11618,11619,11620,11627,11630,11633 and 11636. 8. a peptide species, described polypeptide comprises with SEQ ID NO:6042 to be had＞amino acid sequence of 80% sequence homogeny, and/or comprise the amino acid sequence of the fragment that contains at least 10 continuous amino acids of SEQ ID NO:6042, wherein, described polypeptide is trimeric form.

9. coding is such as the nucleic acid of polypeptide as described in each among the claim 1-8.

10. nucleic acid as claimed in claim 9 comprises and is selected from lower group amino acid sequence: SEQ ID NOS:7191,7273,7275,7277,7279,7281,7283,7285,7287,7289,7291,7292,7293,9968,10066,10084,10299,10505,11323,11563,11639 and 11640.

11. one kind comprises with claim 9 or the described nucleic acid of claim 10 and has＞polynucleotides of the nucleotide sequence of 80% sequence homogeny.

12. polynucleotides that contain the fragment of claim 9 or at least 10 continuous nucleotides of the described nucleic acid of claim 10.

13. can identify such as the antibody of polypeptide as described in each among the claim 1-8.

14. antibody as claimed in claim 13, wherein, described antibody capable identification contains the amino acid sequence of SEQ ID NO:6042 or the polypeptide of its fragment.

15. antibody as claimed in claim 14, wherein, the identification of described antibody capable contains the polypeptide of the trimeric form of the amino acid sequence of SEQ ID NO:6042 or its fragment.

16. antibody as claimed in claim 13, wherein, described antibody be monoclonal antibody,

17. antibody as claimed in claim 13, wherein, described antibody is people's antibody.

18. the immunoassay of SARS virus antigen comprises making the contacted step of each described antibody among sample and the claim 13-17 in the test sample.

19. the immunoassay of the antibody of anti-SARS virus antigen comprises making the contacted step of each described polypeptide among sample and the claim 1-8 in the test sample.

20. the method for the antibody of anti-SARS virus antigen in the test sample, if comprise making described sample and each described polypeptide among the claim 1-8 be combined and contact under the condition of described antibody of existence being fit to described polypeptide, and detect the combination of described polypeptide and described antibody.

21. the method for SARS virus antigen in the test sample, if comprise making described sample and each described antibody among the claim 13-17 be combined and contact under the condition of described antigen of existence being fit to described antibody, and detect the combination of described antibody and described antigen.

22. the vaccine for the treatment of or preventing severe acute respiratory syndrome (SARS) comprises the SARS virus of the SARS virus of deactivation, the SARS virus that kills, attenuation, the SARS virus goods of cracking or the SARS viral antigen of at least a purifying.

23. vaccine as claimed in claim 22 comprises the polypeptide such as each described purifying among the claim 1-8.

24. such as claim 22 or the described vaccine of claim 23, wherein, described antigen is the SARS virus antigen of the purifying of VLP form.

25. such as each described vaccine among the claim 22-24, also comprise adjuvant.

26. vaccine as claimed in claim 25, wherein, described adjuvant is aluminium salt or MF59.

27. such as each described vaccine among the claim 22-26, comprise more than one SARS virus antigen.

28. vaccine as claimed in claim 27, wherein, described antigen is selected from S, E, N and M.

29. vaccine as claimed in claim 22 comprises deactivation of SARS virus.

30. vaccine as claimed in claim 29, wherein, described virus is by chemistry or physical method deactivation.

31. vaccine as claimed in claim 30, wherein, described deactivation comprises with one or more agent treatment that are selected from lower group of effective dose viral: detergent, formaldehyde, formalin, beta-propiolactone and UV light.

32. vaccine as claimed in claim 30, wherein, described deactivation comprises with one or more agent treatment that are selected from lower group of effective dose viral: methylene blue, psoralen and carboxyl fullerene (C60).

33. vaccine as claimed in claim 30, wherein, described deactivation comprises with one or more agent treatment that are selected from lower group of effective dose viral: binary ethamine, acetyl group ethylene imine and radiation gamma.

34. vaccine as claimed in claim 31, wherein, described deactivation comprises with beta-propiolactone to be processed.

35. vaccine as claimed in claim 34, wherein, described beta-propiolactone uses with the concentration of 0.01-0.5%.

36. vaccine as claimed in claim 34, wherein, described beta-propiolactone uses with the concentration of 0.5-0.2%.

37. vaccine as claimed in claim 34, wherein, described beta-propiolactone uses with the concentration of 0.025-0.1%.

38. the method for a deactivation of SARS virus comprises making virus expose 12-24 hour in inactivator under refrigerated storage temperature that the temperature that then raises was hydrolyzed any remaining inactivator in 3 hours.

39. method as claimed in claim 38, wherein, described inactivator is beta-propiolactone.

40. method as claimed in claim 38, wherein, described refrigerated storage temperature is between 0 ℃ and 8 ℃.

41. method as claimed in claim 38, wherein, the temperature of described rising is between 33 ℃ and 41 ℃.

42. a method of making the SARS inactivated vaccine comprises:

A. in mammalian cell cultures, inoculate SARS virus;

B. cultivate infected cell;

C. results contain the supernatant of SARS virus;

D. deactivation of SARS virus; With

E. the SARS virus of purifying deactivation.

43. method as claimed in claim 42, wherein, described mammalian cell cultures is selected from lower group cell type derived from one or more: fibroblast, endothelial cell, liver cell, keratinocyte, immunocyte, mammary glandular cell, smooth muscle cell, melanocyte, nerve cell, prostatic cell, nephrocyte, bone cells, liver cell, retinoblast and stroma cell.

44. method as claimed in claim 42, wherein, described mammalian cell cultures is derived from the cell culture that is selected from lower group: people's cell, non-human primates cell, HeLa cell, human diploid cell, rhesus macaque tire pneumonocyte, HEKC, Vero cell, horse cell, ox cell, sheep cell, dog cell, cat cell or rodent cells.

45. method as claimed in claim 42, wherein, described mammalian cell cultures is derived from Vero cell or fetal rhesus kidney cell.

46. method as claimed in claim 42, wherein, described mammalian cell is cultivated in serum free medium.

47. method as claimed in claim 42, wherein, described mammalian cell is cultivated in protein-free culture.

48. method as claimed in claim 42, wherein, described inoculation step comprises makes SARS virus invest cell culture 60-300 minute.

49. method as claimed in claim 42, wherein, described inoculation step carries out at 25-40 ℃.

50. method as claimed in claim 42, wherein, described purification step comprises that one or more are selected from lower group processing: gradient centrifugation, ultracentrifugation, continuous-flow ultracentrifugation, chromatography, polyethylene glycol precipitation and ammonium sulfate precipitation.

51. method as claimed in claim 42, wherein, described purification step comprises one or more processing that is selected from lower group: ultrafiltration and diafiltration.

52. method as claimed in claim 50, wherein, described Image processing comprises that one or more are selected from lower group Image processing: ion-exchange chromatography, size exclusion chromatography and liquid phase affinity chromatography.

53. method as claimed in claim 52, wherein, described Image processing comprises use, and one or more are selected from lower group chromatographic resin: resin anion (R.A.) and resin cation.

54. method as claimed in claim 52, wherein, described ion-exchange chromatography is processed and is comprised the first step that uses reinforcing yin essence ion exchange resin and the second step that uses strong cation-exchanging resin.

55. method as claimed in claim 50, wherein, described gradient centrifugation purification step comprises density gradient centrifugation.

56. method as claimed in claim 42, wherein, described purification step comprises first step chromatographic purifying and second step gradient centrifugation.

57. method as claimed in claim 56, wherein, described first step chromatographic purifying comprises the liquid phase affinity chromatography.

58. method as claimed in claim 56, wherein, described second step gradient centrifugation comprises density gradient centrifugation.

59. single stranded oligonucleotide that comprises the nucleotide sequence that is selected from lower group: SEQ ID NOS:21-6020,6076-6568,6586-6587,7292-7301,7325-7328,7332-7352,7353-7385,10235-10298,10352-10504,10580-11322 and 11325-11551.

60. single stranded oligonucleotide that comprises the complementary series of the described oligonucleotides of claim 59.

61. such as claim 59 or the described oligonucleotides of claim 60, it comprises 10-30 nucleotides.

62. oligonucleotides as claimed in claim 61 comprises the nucleotide sequence of SEQ ID NO:7292, SEQ ID NO:7293, the complementary series of SEQ ID NO:7292 or the complementary series of SEQ ID NO:7293.

63. the kit of the primer of contained template sequence in the SARS virus nucleic acid target that contains to increase, this kit comprises the first primer and the second primer, wherein said the first primer contains and the basic complementary sequence of the part of described template sequence, and described the second primer contains and the basic complementary sequence of the part of the complementary series of described template sequence, wherein, the interior basic complementary sequence of described primer has been determined two ends of template sequence to be amplified.

64. such as the described kit of claim 63, wherein, described template sequence is contained among SEQ ID NO:1 and/or the SEQ ID NO:2.

65. such as claim 63 or the described kit of claim 64, wherein, described the first primer comprises 8 of SEQ ID NO:1 or the fragment of polynucleotides more, the second primer comprises 8 of SEQ ID NO:1 complementary series or the fragment of polynucleotides more.

66. such as claim 63 or the described kit of claim 64, wherein, described the first primer comprises 8 of SEQ ID NO:2 or the fragment of polynucleotides more, the second primer comprises 8 of SEQ ID NO:2 complementary series or the fragment of polynucleotides more.

67. such as the described kit of claim 63, wherein, described the first primer is each described oligonucleotides among the claim 59-62, the second primer is each described oligonucleotides among the claim 59-62.

68. such as each described kit among the claim 63-67, the probe that also comprises mark, described probe comprises SEQ ID NO:1 and/or SEQ ID NO:2 8 or the fragment of polynucleotides more, or the complementary series of described fragment, and this fragment is positioned at described template sequence.

69. such as each described kit among the claim 63-68, wherein, described the first primer and/or the second primer comprise and are selected from lower group nucleotide sequence: SEQ ID NOS:21-6020,6076-6568,6586-6587,7292-7301,7325-7328,7332-7352,7353-7385,10235-10298,10352-10504,10580-11322 and 11325-11551.

70. such as each described kit among the claim 63-68, wherein, described the first primer and/or the second primer comprise the complementary series of the nucleotide sequence that is selected from lower group: SEQ ID NOS:21-6020,6076-6568,6586-6587,7292-7301,7325-7328,7332-7352,7353-7385,10235-10298,10352-10504,10580-11322 and 11325-11551.

71. whether there is the method for SARS virus in the test sample, the method comprises providing suspects the sample that contains SARS viral nucleic acid target, with the contained template sequence in the described SARS virus nucleic acid target that increases of each described kit among the claim 63-70, and the template sequence of detection amplification, wherein, exist the template sequence that increases to show in the described sample and have SARS virus.

72. such as the described method of claim 71, wherein, described amplification is finished with following methods: the amplification of PCR, transcriptive intermediate, reverse transcription PCR, ligase chain reaction, strand displacement amplification or based on the amplification of nucleotide sequence.

73. the double stranded rna molecule of the sars coronavirus of a length about 10 to about 30 nucleotides in can the deactivation mammalian cell.

74. such as the described double-stranded RNA of claim 73, wherein, the sequence of a chain is identical with target sequence at least 90%, wherein said target sequence is the fragment of SEQ ID NO:1 and/or SEQ ID NO:2.

75. such as claim 73 or the described double-stranded RNA of claim 74, wherein, described target sequence comprises and is selected from lower group nucleotide sequence: SEQ ID NOS:7292,7293,7294,7295,7296,7297,7298,7299,7300 and 7301.

76. such as each described double-stranded RNA among the claim 73-75, it comprises the nucleotides of at least one modification.

77. a method for the treatment of SARS patient comprises the molecule less than 1000g/mol that gives this patient treatment effective dose.

78. such as the described method of claim 77, wherein, described molecule has the armaticity zone and is selected from the hetero atom of O, S or N more than one.

79. a method for the treatment of SARS patient comprises the compound of group under being selected from that gives this patient treatment effective dose: the inhibitor of nucleoside analog, class peptide, oligopeptides, polypeptide, protease inhibitors, 3C-sample protease inhibitors, papain-sample protease inhibitors or RNA-directed RNA polymerase.

80. comprising, a method for the treatment of SARS patient, the method give this patient's SAID and at least a antiviral compound.

81. a method for the treatment of SARS patient, the method comprises the compound of group under being selected from that gives the patient treatment effective dose: acyclovir, gancyclovir, vidarabidine, foscamet, cidofvoir, amantidine, Ribavirin, trifluorothymidine, Zidovudine, Didanosine, zalcitabine, the listed antiviral compound of table 1, the listed antiviral compound of table 2 or interferon.

82. such as the described method of claim 81, wherein, described interferon is interferon-' alpha ' or interferon-beta.

83. such as each described method among the claim 77-82, wherein, described molecule or compound are carried by sucking.

84. a method of identifying the therapeutic activity agent, the method may further comprise the steps: (a) make therapeutic activity agent and the cells contacting that is infected by SARS virus; (b) measure the attenuation situation of the enzyme relevant with SARS.

85. viral vectors or particle that is used for carrying in the body claim 9 or the described nucleic acid of claim 10.

86. such as the described viral vectors of claim 85, wherein, described carrier is adenovirus vector, poxvirus vector or α viral vectors.

87. sub-particle of α virus replication that contains one or more SARS virus antigens.

88. such as the described replicon particle of claim 87, wherein, described SARS virus antigen is spike protein.

89. such as the described replicon particle of claim 87, wherein, described particle comprises the replicon derived from Venezuelan equine encephalitis (VEE) virus, also comprises the coating derived from sindbis virus (SIN) or Semliki forest virus (SFV).

90. vaccine that contains one or more SARS virus antigens and one or more Respirovirus antigens.

91. such as the described vaccine of claim 90, wherein, described Respirovirus antigen is selected from lower group: influenza virus, ERC group virus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus(RSV) (RSV), adenovirus, super Pneumovirinae and rhinovirus.

92. such as the described vaccine of claim 91, wherein, described Respirovirus antigen is from influenza virus.

93. such as the described vaccine of claim 90, wherein, described Respirovirus antigen is from the coronavirus outside the SARS virus.

94. polypeptide that comprises the immunogenic fragments that is exposed to the surface of amino acid sequence SEQ ID NO:6042.

95. such as the described polypeptide of claim 94, wherein, described fragment does not comprise terminal last 50 amino acid of SEQ ID NO:6042 C-.

96. such as the described polypeptide of claim 94, wherein, described fragment does not comprise the membrane spaning domain of SEQ ID NO:6042.

97. such as the described polypeptide of claim 94, wherein, described fragment does not comprise the terminal cytoplasmic structure of the C-territory of SEQ ID NO:6042.

98. such as the described polypeptide of claim 94, wherein, described fragment does not comprise the N-terminus signal sequence.

99. one kind comprises the polynucleotides that separate that are selected from SEQ ID NOS:9968 and 10066 nucleotide sequence.

100. such as the described polynucleotides of claim 99, wherein, described polynucleotides comprise with the polynucleotide sequence that is selected from SEQ ID NOS:9968 and 10066 to be had＞nucleotide sequence of 80% sequence homogeny.

101. polynucleotides that separate that comprise the fragment that is selected from SEQ ID NOS:9968 and at least 15 continuous nucleic acid of nucleotide sequence of 10066, wherein, described fragment does not contain whole SEQ ID NO:10033.

102. polypeptide that contains by the separation of the amino acid sequence of each described sequential coding among the claim 99-101.

103. such as the described polypeptide of claim 102, it comprises and is selected from lower group amino acid sequence: SEQ ID NOS:9969-10032,10067 and 10015.

104. such as the described polypeptide of claim 103, wherein, described amino acid sequence is selected from: SEQ ID NOS:9997,9998 and 10015.

105. the expression construction of a recombinant expressed SARS virus spike protein, wherein, described construction comprises the nucleotide sequence that is selected from SEQ ID NOS:6578-6583.

106. the mammal cell line of a stably express SARS virus antigen.

107. such as the described clone of claim 106, wherein, described clone is Chinese hamster ovary (CHO) cell.

108. such as the described clone of claim 106, wherein, described SARS virus antigen is spike protein or its fragment.

109. such as the described clone of claim 106, wherein, described spike protein be brachymemma to remove the cross-film sequence.

110. a method of identifying the therapeutic activity agent, the method may further comprise the steps: the therapeutic activity agent is contacted with the buffer solution that contains the SARS enzyme; (b) the attenuation situation of mensuration SARS enzyme.

111. such as the described method of claim 110, wherein the SARS enzyme is SARS protease.

112. such as the described method of claim 111, wherein, described buffer solution also comprises the peptide with SARS protease cutting site.

113. such as the described method of claim 110, wherein, described mensuration is finished by fluoremetry.

114. such as each described vaccine among claim 22-37 and the 90-93, also comprise adjuvant.

115. such as the described vaccine of claim 114, wherein, described adjuvant is SMIP.

116. such as the described vaccine of claim 115; wherein; described SMIP compound is selected from: acyl piperazine, couroupitine A, indole dione, tetrahydroisoquinoline, benzo ring diketone, amino azepine vinyl compound, thiosemicarbazone, lactams, aminobenzimidazole quinolinone, hydroxyl phthalimide, benzophenone, isoxazole, sterol, quinazolone, pyrroles (pyrole), anthraquinone, quinoxaline, triazine, indoles and pyrazolopyrimidine, or their pharmaceutically acceptable salt, ester or prodrug.

117. give the vaccinated method of object, comprise giving each described vaccine among this object such as claim 22-37 and the 90-93 for one kind.

118. such as the described method of claim 117, also comprise giving SMIP.

119. the method such as each described treatment patient among the claim 77-82 also comprises giving at least a SMIP compound.

120. the method such as each described treatment patient among the claim 77-82 also comprises giving at least a SMIS compound.

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