CN101039955A - Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein - Google Patents

Abstract

Nucleic acid molecules, polypeptides, immunogenic compositions, vaccines, and methods of making and using the nucleotides and encoded polypeptides associated with the Spike protein of SARS Corona Virus (SARS CoV) are disclosed.

Description

The nucleic acid relevant, polypeptide, expression method and immunogenic composition with sars cov spike protein

Related application

It is the U.S. Patent application No.10/860 on June 4th, 2004 that the application requires the applying date, 641 and the applying date be the U.S. Provisional Patent Application No.60/578 on June 10th, 2004,348 right of priority is incorporated above-mentioned application into the application by reference at this.On May 23rd, 2005 submitted to U.S. Patent application No.10/860, and 641 change to the request of provisional application.

Technical field

The present invention relates to purifying with isolating nucleic acid, polypeptide, purifying and nucleic acid isolated polypeptide, this type of polypeptide of encoding, produce the method for this type of polypeptide of recombinant forms, at antibody and this type of nucleic acid and the purposes of polypeptide in diagnostic method, test kit, immunogenic composition, vaccine or antiviral therapy that these polypeptide produced.

Background technology

2002, a kind of new infectious diseases appearred in the Guangdong Province of southern china, was called severe acute respiratory syndrome (SARS).SARS propagates in 29 countries, it is reported that 8,098 people get involved, and causes 774 people's death (Stadler et al.).Though contained the SARS epidemic situation, and do not known that when or SARS will occur once more in the crowd by the Isolation Quarantine measure of strictness.

The principal character of SARS is a flu syndrome, comprises that high heat surpasses to invade profit (Stadler et al.) on 100.4 , myalgia, dry cough expiratory dyspnea, low lymphocyte mass formed by blood stasis and the chest x-ray.Have 38% in all cases because of having occurred causing the pneumonia of acute respiration obstacle to need the breathing apparatus.Overall mortality rate is approximately 10%, but changes obviously with the difference at age, as if because SARS is lighter in children crowd, but the mortality ratio in the older is then up to 50%.

What cause SARS is by coronavirus (CoV) unknown before a kind of, and coronavirus is diversified big, an envelope virus group, causes respiratory tract and intestinal tract disease in humans and animals.SARS CoV separates from FRhK-4 cell and the Vero E6 cell inoculated from patient's clinical samples, and viewed those symptoms in the human case of SARS have appearred being similar in the macaque that has inoculated this virus.Up to now, separated and checked order and surpass 30 kinds of different SARSCoV.

SARS CoV contains the rna gene group (Accession No.AY310120) of about 30kB, and has the many typical feature of coronavirus.Nucleotide 1-72 contains the RNA leader sequence of inferring, its be positioned at cross over 192 Nucleotide non-translational region (UTR) before.What be positioned at the UTR downstream is two eclipsed open reading-frame (ORF)s, and they have covered about 2/3 genome (Nucleotide 265-21485), and proteins encoded enzyme and duplicate and transcribe required protein (summary can referring to Stadler et al., 2004).The remaining institute 3 of genome ' part 4 kinds of structural protein of encoding, these albumen in all CoV all with identical series arrangement: furcella (Spike) albumen, envelope protein, membrane glycoprotein and nucleocapsid protein.Also encode and be called the extra Nonstructural Protein of " subsidiary gene (accessory gene) " in the genomic structural protein of SARS CoV zone.Although the genomic Overall Organization Structure of SARS CoV is similar to other coronavirus, coded proteinic amino acid conservative property is lower usually.

Spike protein forms the peculiar big surperficial projection of coronavirus.Furcella is had 1,255 amino acid by a large amount of glycosylations, and containing what be positioned at N-terminal spherical head, next-door neighbour is stem, single film district and the short endochylema afterbody (referring to Stadler et al.) of striding.

Although being arranged, report thinks that beta-interferon the duplicating of external interference SARS virus, also do not ratify any medicine or vaccine now.In addition, the work of extensive examination potential replication inhibitors does not also have achievement at present in existing antiviral agent or big chemical agent library.In fact, it also almost is impossible will making a definite diagnosis SARS when head examines, because the susceptibility of existing detection method and specificity can be along with coming in contact or the time lapse after the symptom occurring and change (referring to Raineret al.).Also can in first week of morbidity, diagnose SARS at present, also not have to provide in a few hours after collected specimens result's method without any easy, accurate detection method.In view of these reasons, very need further detail knowledge SARS CoV albumen.Such understanding can or be controlled this infection for treatment effective means are provided, and helps the mankind's SARSCoV infection is diagnosed.

Summary of the invention

Therefore, the objective of the invention is to satisfy the demand of the prior art.The present invention includes a kind of nucleic acid molecule of purifying, it comprises the dna sequence dna of SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:6.The present invention also comprises and above-mentioned sequence complementary nucleic acid molecule, for example complementary sequence of total length.

The present invention includes the nucleic acid molecule of purifying of the aminoacid sequence of the double chain acid molecule of those dna sequence dnas that comprise SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:6 and coding SEQ ID NO:4 or SEQ ID NO:7.The present invention had both comprised the RNA and the DNA nucleic acid molecule that also comprise two strands of strand.These molecules can be used for detecting the included strand of the present invention and double-stranded RNA and DNA.Double chain DNA probe can detect the arbitrary chain equivalent nucleic acid molecule with described nucleic acid molecule.

Those under high stringency with the double-stranded DNA of the sex change of the dna sequence dna that comprises SEQ ID NO:2, SEQ ID NO:3 or SEQID NO:6 or with the nucleic acid molecule of the purifying of the making nucleic acid molecular hybridization of the purifying of the aminoacid sequence of coding SEQ ID NO:4 or SEQ ID NO:7, comprise within the scope of the invention.

The present invention also comprises by vitro mutagenesis from the nucleic acid molecule of SEQ ID NO:1-3 and 6 deutero-purifying.Vitro mutagenesis comprises multiple technologies known in the art, includes but not limited to, site-directed mutagenesis, random mutagenesis and external nucleic acid are synthetic.

Nucleic acid molecule of the present invention comprises DNA and RNA, these nucleic acid molecule are called " furcella nucleic acid (Spike nucleic acids) " or " furcella DNA (Spike DNA) " at this, are called " furcella polypeptide " or " spike protein " by the aminoacid sequence of these molecule encodings at this.

The present invention also comprises the nucleic acid molecule of the purifying that obtains from SEQ ID NO:1-3 and 6 degeneracys because of the genetic code degeneracy, as the nucleic acid molecule of the purifying of the homologue of the allele variant of furcella nucleic acid or furcella nucleic acid.

The present invention includes the nucleic acid of comparing the purifying that demonstrates the expression of enhanced spike protein with SEQ ID NO:1.

The present invention also comprises the nucleic acid of comparing the purifying that demonstrates the expression of enhanced spike protein with SEQ ID NO:1, and wherein at least a negative cis acting signal is replaced, and this does not change coded proteinic sequence.The negative cis acting signal that the present invention includes includes but not limited to be rich in RNA unstable motif, tumor-necrosis factor glycoproteins, secondary tract (secondary stretch), donor splicing site and acceptor site and inner poly (A) site of AU.

The present invention also comprises the nucleic acid molecule of comparing the purifying that demonstrates the expression of enhanced spike protein with SEQ ID NO:1, wherein strengthens expression by increasing the expression enhancement sequences.Express enhancement sequences and include but not limited to the Kozak consensus sequence of initial ATG upstream and extra STOP codon.

Those skilled in the art can understand how the Kozak consensus sequence is placed suitable position based on prior art.

The present invention also comprises the nucleic acid molecule of comparing the purifying that demonstrates the expression of enhanced spike protein with SEQ ID NO:1, and wherein the use of codon is optimized according to the preference of Chinese hamster (Cricetulus griseus).

The present invention also comprises the nucleic acid molecule of comparing the purifying that demonstrates the expression of enhanced spike protein with SEQ ID NO:1, the part of the coding spike protein of the nucleic acid molecule of wherein said purifying is compared with SEQID NO:1, its GC percentage composition increase is about at least 10%, and has wherein avoided occurring the zone of GC content high (＞80%) or extremely low (＜30%) under possible situation.

The present invention also comprises the nucleic acid molecule of comparing the purifying that demonstrates the expression of enhanced spike protein with SEQ ID NO:1, and the displacement of wherein said at least a negative cis acting signal and wherein said at least a extra expression enhancement sequences do not comprise: inner TATA box, chi site and ribosome entry site(RES); Be rich in AT or be rich in the tract (sequence stretches) of GC; Tumor-necrosis factor glycoproteins and RNA secondary structure; With donor splicing site and acceptor site.

The present invention also comprises the polypeptide by the purifying of these nucleic acid molecule encodings, comprises the glycosylation and the non-glycosylated form of the polypeptide of described purifying.

The present invention also comprises and instructs recombinant vectors that these nucleic acid molecule express and with the host cell of these carriers conversions or transfection.

The present invention includes polyclone or monoclonal antibody with furcella polypeptide bonded purifying, for example neutralizing antibody.

The present invention also comprises the method for preparing the furcella polypeptide, is included under the condition that promotes to express and cultivates host cell, and collect described polypeptide in substratum.The present invention especially is included in and expresses the furcella polypeptide in the zooblast.

The present invention also comprises the furcella polypeptide of mark.Preferably, the polypeptide of described mark is the form of purifying.Equally preferably, it is other that described polypeptide unlabelled or mark can be contained people's the humoral immunization knowledge of anti-furcella polypeptide antibody.Described polypeptide can carry out mark with the immunoassay marker that for example is selected from next group: radioactively labelled substance, enzyme marker, fluorescent marker, chemiluminescent labels and chromophoric group.

The present invention also provide furcella polypeptide of the present invention and identification described polypeptide antibody between immunocomplex.Described immunocomplex can carry out mark with the immunoassay marker that is selected from next group: radioactively labelled substance, enzyme marker, fluorescent marker, chemiluminescent labels and chromophoric group.

In addition, the invention provides the method that is used to detect SARS CoV infection.Described method comprises providing and comprises the doubtful composition that has infected the biologic material of SARS CoV, and measures the existence of SARSCoV furcella polypeptide.Described polypeptide is normally by electrophoresis or by using the immunoassay that has the antibody of immunological response with furcella polypeptide of the present invention to measure.

The present invention also provides the in-vitro diagnosis method, and it is used to detect the antigenic antibody that whether exists in conjunction with comprising furcella polypeptide of the present invention.Described method comprises described antigen is contacted with biological fluid that the time of contact and condition are enough to make that the antibody in described antigen and the biological fluid forms immune complex, detect the formation of described mixture then.Detect step and can further comprise the formation of measuring described immune complex.Preferably by measure the formation of immune complex based on the immunoassay of western blotting technique, ELISA (enzyme-linked immunosorbent assay), FACS, indirect immunofluorescence assay method or immunoprecipitation assay.

The present invention also comprises diagnostic kit, and whether it is used for detecting and exists and furcella polypeptide bonded antibody of the present invention, and described test kit contains the antigen that comprises described furcella polypeptide, and is used to detect the reagent that forms immunocomplex between described antigen and the antibody.The amount of wherein said antigen and described reagent is enough to carry out described detection.

The present invention also provides a kind of immunogenic composition, and it comprises furcella polypeptide of the present invention or its mixture and medicinal acceptable carrier, and the amount of described furcella polypeptide or its mixture is enough to induction of immunity originality or protective response in vivo.Immunogenic composition can contain the Alum adjuvant.Vaccine composition of the present invention comprises the furcella polypeptide and the medicinal acceptable carrier of dosis neutralisata.

Therefore, polypeptide of the present invention can be used as the part of diagnosis composition, whether has the antibody that resists the antigen protein relevant with SARS CoV to detect.

In addition, the furcella polypeptide also can be used for producing the antibody whether detection exists the antigen protein relevant with SARS CoV.

Polypeptide of the present invention also can be used for producing neutralizing antibody, these neutralizing antibodies or inactivation of viruses, or reduce virus vigor in vivo, or the duplicating of inhibition or blocking virus.So that when B cell branch that activate immunity is replied in receptor's host or inducing cytotoxic T lymphocyte responses (CTL), the ability of this initiation virucidin is a particularly important when polypeptide of the present invention being used for immune composition or preventive vaccination composition.

The invention still further relates to the vaccine composition that is used for people and Mammals are carried out anti-SARS CoV immunization, it comprises above-mentioned immunogenic composition and makes up one or more medicinal acceptable vehicle (for example brine buffer solution), and randomly make up at least a adjuvant, aluminium hydroxide or belong to the compound of muramylpeptides family for example.

The present invention also comprises and is used to detect the method that whether has SARS CoV, comprising:

(1) sample of the viral genetic of the doubtful SARS of containing CoV is contacted with at least a nucleotide probe and

(2) hybridization between the viral genetic in described nucleotide probe of detection and the described sample,

Wherein said nucleotide probe is complementary to the full length sequence of the furcella nucleic acid of purifying of the present invention.

Other features of the present invention and advantage, its partial content will be set forth in the following description, and partial content will be conspicuous based on specification sheets, perhaps can learn by putting into practice the present invention.Purpose of the present invention and superior part can be by realizing as those pointed in appended claims elements and combination or obtaining.

No matter should be understood that, be the generality description or the detailed description subsequently of front, all only is used for example and explains the present invention, and have no intention claimed invention is limited.

Description of drawings

Appended accompanying drawing is the part of book as an illustration, and it for example understands some embodiments of the present invention, and these accompanying drawings and text description are used to explain principle of the present invention jointly.

Figure 1 shows that the expression of Spike-HKU-PRC in the 293T of transfection cell.Swimming lane 1 representative is with the pcDNA-Spike-Pasteur cells transfected, swimming lane 2 representatives are with the pcDNA-HKU-PRC cells transfected, swimming lane 3 representatives are with the SFV-Spike-Pasteur-modif cells transfected, swimming lane 4 is empty, and swimming lane 5 representatives are from the furcella (Spike) of the purifying of the bhk cell of transfection.

Figure 2 shows that to be used for SARSCoV spike protein sequence RNA and protein immunization, that have the Flag peptide sequence (SEQ ID NO:5), with the single-letter abbreviation expression of standard.Dash area is the sequence corresponding to SARS CoV spike protein, and underscore partly is the sequence that comprises the Flag peptide.This representative sequence is expressed in Semliki Forest Virus (SFV) carrier.

Fig. 3 is that SFV-Spike pulse-append formula (pulse-chase) infected B HK cell with mark is through the SDS-PAGE of M2 (Flag) antibody mediated immunity post precipitation figure.Fixed time point after appending is collected cell." * " represents high mannose N-glycan EndoH susceptibility furcella, and " O " represents compound N-glycan EndoH resistivity furcella, and the deglycosylated furcella of " # " high mannose N-glycan EndoH susceptibility.

Figure 4 shows that the expression of the Spike on the bhk cell plasma membrane that has infected SFV Spike.With M2 antibody spike protein is carried out mark, with the Erp72 monoclonal antibody endoplasmic reticulum (ER) is dyeed simultaneously.

Fig. 5 is the Western engram analysis, and it shows SARS CoV protein binding sACE2 acceptor.To carry out incubation and carry out sds gel electrophoresis with sACE2 with the M2 pearl of Spike (swimming lane 1 and 4) bag quilt or M2 pearl (swimming lane 3 and 6) in contrast, then with anti-ACE2 antibody or carry out the Western trace with Mab M2 in contrast with BAP bag quilt.Although both existed Spike also to have BAP albumen (swimming lane 4 and 6) in the reaction, Spike only arranged in conjunction with ACE2 (swimming lane 1 and 3).

Fig. 6 shows the antibody that carries out the anti-reorganization of the mouse generation Spike of immunization with the SARS CoV spike protein of the immune purifying of reorganization.Use the mixed mice serum (n=5) of dilution in 1: 100 to carry out the Western trace.Swimming lane 1 and 2 is represented respectively and is used from control animal (CTRL) and inoculate the Western trace that the preimmune serum of the animal (VACC) of Spike carries out. Swimming lane 3 and 4 is represented the Western trace that uses the 34th day serum from CTRL and VACC group to carry out respectively, and swimming lane 5 and 6 represent the Western trace that uses the 42nd day serum organizing from CTRL and VACC to carry out respectively.Swimming lane 7 representative SARS patients' serum, swimming lane 8 representative is a kind of commercial from the serum (dilution in 1: 50) with the rabbit of spike protein immunity.

Fig. 7 shows the antibody that carries out the anti-reorganization of the mouse generation Spike of immunization with the SARS CoV spike protein of the immune purifying of reorganization.Use the mixed mice serum (n=5) of dilution in 1: 50 to carry out facs analysis.The right side is SARS patients serum in contrast.

Fig. 8 shows that mouse that the SARS CoV spike protein with the immune purifying of reorganization carries out immunization produces the antibody of anti-SARS CoV.Use the mixed mice serum (n=5) of dilution in 1: 50 on FRHK4 cell that has infected SARS CoV or the false FRHK4 cell that infects, to carry out immunofluorescence analysis.

Fig. 9 (A), 9 (B) and 9 (C) have shown the nucleotide sequence (SEQ IDNO:1) of Spike-Pasteur.Each Spe I site indicates with underscore, and what shade indicated is to replace to form the nucleic acid residue of Spike-Pasteur-modif.

Figure 10 (A), 10 (B) and 10 (C) have shown the nucleotide sequence (SEQ ID NO:2) of Spike-Pasteur-modif.The sudden change that shade indicates causes Spe I site to eliminate in Spike-Pasteur.

Figure 11 (A), 11 (B), 11 (C), 11 (D), 11 (E) and 11 (F) have shown nucleotide sequence (SEQ ID NO:3) and the complementary strand thereof of Spike-HKU-PRC.The nucleic acid sequence encoding furcella polypeptide that shade indicates.Figure 11 (A), 11 (B), 11 (C), 11 (D), 11 (E) and 11 (F) have also shown the aminoacid sequence (SEQ ID NO:4) of the Spike that is blended in the Flag peptide.The STOP codon indicates with asterisk.

Figure 12 (A) and 12 (B) have shown the nucleotide sequence (SEQ ID NO:6) of the optimization of the encoding SARS CoV furcella polypeptide in the Spike-HKU-PRC.The difference of SEQ ID NO:6 and SEQ ID NO:3 is that it does not contain the sequence of coding Flag peptide or upstream or downstream sequence.

Shown in Figure 13 is sequence by the SARS CoV furcella polypeptide (SEQID NO:7) of Spike-HKU-PRC coding.

Figure 14 shows that plasmid of the present invention, i.e. the 040078pPCR-Script of mark, it contains the sequence of the Spike-HKU-PRC that encodes.Be assembled into synthetic gene 040078 from the synthetic oligonucleotide.By KpnI and SacI restriction site with this fragment cloning go into pPCR-Script Amp (Stratagene, LaJolla, CA, USA).

Figure 15 shows that plasmid of the present invention, promptly the 040086pcDNA3.1 (+) of mark is also referred to as 040078pcDNA3.1 (+), and it contains the sequence of the coding Spike-HKU-PRC of the BamHI restriction site that is cloned into pcDNA3.1 (Invitrogen).

Figure 16 shows that the purity of the spike protein that is used for immunization, and shown that silver dyes painted SDS-PAGE gel.Sample comprises: (M) molecular weight standard; (1) 720ng S albumen; (2) 360ng S albumen; (3) 180ng S albumen; (4) 90ng S albumen.Indicated molecular weight, arrow is depicted as the position (upper arrow) of complex glycosylation monomer spike protein and the position (below arrow) of high mannose monomer spike protein.

Figure 17 is presented at serum antibody response in the animal of TriSpike+Alum immunity and strengthens.Analyzed from serum of the mouse of immunization and the reactivity of TriSpike.(A) contrast is in SARS patient's the high titre and antiserum(antisera), the rabbit anteserum of anti-S1 and the M2 monoclonal antibody of anti-FLAG peptide.To from adding Alum adjuvant (group A) with TriSpike or carrying out the Western engram analysis with the mixed serum that TriSpike does not add Alum adjuvant (group B) mice immunized (n=3).Collect serum and, be used for the Western engram analysis at specified time point with dilution in 1: 1000.All serum all react with the antibody of assessment at the FLAG label with the reference protein (BAP-FLAG) of FLAG mark and produce.The IgG polyclonal antibody of goat anti-mouse, people or rabbit that use HRP puts together detects immunocomplex.(B) the neutralization activity that infects from the anti-SARS CoV of the serum of the mouse of immunization at analyzed in vitro with the FRhk4 cell.Neutralization activity (from the 49th day to the 87th day) from the serum of the mouse that has only inoculated TriSpike reduces rapidly, but keeps stablizing in the same reduced time from the neutralization activity of the serum of the mouse that has inoculated TriSpike+Alum.

Figure 18 demonstration is replied with TriSpike+Alum immunized mice mucosa immunity-inducing.Collect from the movement and the nose irrigating solution sample of immune mouse (TriSpike or TriSpike+Alum) and analyze it and the reactivity of TriSpike (A-B).With the M2 monoclonal antibody of anti-FLAG peptide in contrast.(A) collect the faecal samples of immunized mice and dilute according to 1: 500 at the 44th day, be used for the Western engram analysis.Goat anti-mouse IgG or the IgA polyclonal antibody puted together with HRP detect immunocomplex.(B) be depicted as and (A) essentially identical experiment, difference only is to carry out the Western engram analysis with the mixing nose irrigating solution sample of collecting from immunized mice at the 65th day.Nose irrigating solution sample according to dilution in 1: 25, is used for the Western engram analysis.(C) collect from the faecal samples of immunized mice, and with the neutralization activity of FRhk4 cell in the anti-SARSCoV infection of analyzed in vitro.Only detecting weak neutralization activity after the immunization for the third time.Analyzed nose irrigating solution sample from immunized mice, but external do not find to can observe in and activity level.

Figure 19 has shown the immunogenicity of TriSpike in Golden Syrian hamster.Analysis from the hamster of the TriSpike+Alum of concentration shown in having inoculated and from the reactivity of the serum of contrast hamster and TriSpike with in and situation.(A) use FACS to analyze immune serum and the natural proteic reactivity of TriSpike.Will from the serum (the 0th, 21 and 42 day) of the hamster of the subcutaneous immunity of TriSpike of 2,10 or 20 μ g according to dilution in 1: 100, with the BHK-21 cell response alive of on plasma membrane, expressing TriSpike.The anti-hamster IgG of the goat polyclonal antibody that uses FITC to put together is differentiated immunocomplex.The result is with MFI (average fluorescent strength) value representation.The MFI value reaches maximum in the immunity back second time (after the dosage 2), and (after the dosage 3) keeps stable after immunity for the third time.(B) the active little neutralization mensuration of the SARS CoV that carries out on the comfortable FRhk4 cell (microneutralization assay) (the 100TCID50/ hole is the end eventually) that gets of neutralization.

Detailed Description Of The Invention

Had been found that to be used for the optimization dna sequence dna that enhanced SAR S CoV spike protein is expressed, comprised Spike-Pasteur-modif (SEQ ID NO:2) and Spike-HKU-PRC (SEQ ID NO:3 and 6).

Nucleotide sequence of the present invention comprises DNA and the RNA sequence of separation, and described DNA and RNA sequence are hybridized with SEQ ID NO:2 described herein, 3 and 6 under medium or high stringency, and coding furcella polypeptide. At this, medium stringency is that those skilled in the art are known and be documented in Sambrook et al.Molecular Cloning:A Laboratory Manual, 2ed.Vol.1, pp.1.101-104, Cold Spring Harbor Laboratory Press, (1989), it comprises nitrocellulose filter use pre-wash solution 5X SSC, 0.5%SDS, 1.0mM EDTA (pH 8.0), hybridization conditions is 50% formamide, 6X SSC, 42EC (perhaps other similar hybridization solutions, for example Stark ' s solution, 50% formamide, 42EC), and wash conditions be about 60EC, 0.5X SSC, 0.1%SDS. High stringency is defined as: hybridization conditions as mentioned above, and wash conditions is 68EC, 0.2XSSC, 0.1%SDS. Those skilled in the art can understand, in case of necessity can according to various factors for example the length of probe adjust the salinity of temperature and wash solution.

By the coded polypeptide of these new nucleic acid referred to here as " furcella polypeptide (Spike polypeptide) " or " spike protein (Spike albumen) ". At this, these terms refer to a class polypeptide, they also comprise the protein of the amino acid sequence with SEQ ID NO:4 or SEQ ID NO:7, and those proteins and peptides with high similarity (at least 90% similitude) of amino acid sequence therewith, and described proteins and peptides has immunoreactivity. In addition, " furcella polypeptide " and " spike protein " also refer under high stringency those protein coded with the nucleic acid molecules of the nucleic acid chains hybridization of the coded sequence complementation of SEQ ID NO:3 or SEQ ID NO:6.

Term " purifying " refers to that at this furcella polypeptide does not combine with other protein or polypeptide basically, for example is the purified product as the recombinant host cell culture, or as the purified product of non-recombinant sources. Term " in fact purifying " refers to a kind of mixture at this, it contains the furcella polypeptide, if not and have the known protein to use specific antibody and remove, it does not combine with other protein or polypeptide basically, and the furcella polypeptide of described in fact purifying can be used as antigen.

Furcella polypeptide " variant " refers to and the natural furcella polypeptide polypeptide of homology in fact at this, but owing to one or more disappearance, insert or replace and have the amino acid sequence that is different from natural furcella polypeptide. The variant amino acid sequence preferably has at least 80% homogeny, more preferably at least 90% homogeny with the amino acid sequence of natural furcella polypeptide. Homogeny percentage is confirmable, for example can use Devereux et al. (Nucl.Acids Res.12:387,1984) 6.0 editions comparative sequences information of described GAP computer program and determine that this program can obtain from University of Wisconsin Genetics Computer Group (UWGCG). The GAP program is used the comparison method of Needleman and Wunsch (J.Mol.Biol.48:443,1970), and Smith and Waterman (Adv.Appl.Math2:482,1981) revise the method. The preferred default parameters of GAP program comprises: (1) is used for the monobasic comparator matrix of nucleotides (identical value is 1, not identical value is 0), and Gribskov and Burgess, Nucl.Acids Res.14:6745,1986 described weighting comparator matrixs, referring to Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358,1979; (2) point penalty of each breach is 3.0, and each symbol in each breach has extra 0.10 point penalty; And (3) terminal breach is without point penalty.

Variant can comprise the sequence that conservative replaces, and the conservative replacement refers to that a kind of given amino acid residue is had the residue replacement of similar biochemical characteristic. The example that conservative replaces comprises aliphatic residue displacement to each other, for example Ile, Val, Leu or Ala replacement each other; Or polar residues displacement each other, for example between Lys and the Arg; Between Glu and the Asp; And between Gln and the Asn. Other these type of preservative replacement are known, for example have the displacement in the whole zone of similar hydrophobic property. The present invention also comprises naturally occurring furcella polypeptide variants. The example of this type of variant is the protein that produces because of substituting mRNA montage event or because of the protease cracking of furcella polypeptide. Be attributable to proteolysed variation comprises the end that for example occurs behind dissimilar host cell inner expressions difference, this is owing to removed due to one or more end amino acid from furcella polypeptide protein hydrolysis. The variation that is attributable to frameshit comprises the difference of the end that for example occurs behind dissimilar host cell inner expressions, this is because due to the different amino acid.

As mentioned above, the invention provides and separate and furcella polypeptide purifying or homogeneous, can be restructuring also can be nonrecombinant. Can be used as the variant of natural furcella polypeptide of antigen and derivative can suddenly change by the nucleotide sequence to the natural furcella polypeptide of encoding and obtain. Can be by the change of multiple conventional method realization to natural acid sequence. By the synthetic oligonucleotides that contains mutant nucleotide sequence, wherein the mutant nucleotide sequence both sides have and can realize the restriction site that is connected with the fragment of native sequences, and introducing specific locus thus will suddenly change. After connecting, the analog that resulting reconstruction sequence coding has amino acid needed insertion, replacement or disappearance.

Perhaps, can provide with the site-specific mutagenesis method that oligonucleotides instructs a kind of gene of change, wherein can change wherein predetermined codon by replacing, lack or inserting. The illustrative methods that realizes above-mentioned change can be referring to Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73,1985); Craik (BioTechniques, January 1985,12-19); Smith et al. (Genetic Engineering:Principles and Methods, Plenum Press, 1981); Kunkel (Proc.Natl.Acad.Sci.USA 82:488,1985); Kunkel et al. (Methods in Enzymol.154:367,1987); With United States Patent (USP) 4,518,584 and 4,737,462, all these documents are all incorporated the application by reference into.

In one aspect of the invention, the furcella polypeptide can be used for preparing the antibody of specific binding furcella polypeptide. Term " antibody " comprises for example binding partners (binding partner) that produces of F (ab ') 2 and Fab fragment and any recombination method of polyclonal antibody, monoclonal antibody, its fragment. If antibody is to be higher than or to equal about 10⁷M ^-1K_aValue is incorporated into the furcella polypeptide, and then described antibody is believed to occur specific binding. Can adopt routine techniques easily to determine the affinity of binding partners or antibody, for example adopt Scatchard et al., Ann.N.YAcad.Sci., the described method of 51:660 (1949). Can adopt the known method of prior art easily to produce polyclonal antibody from multiple source, for example from horse, ox, goat, sheep, dog, chicken, rabbit, mouse or rat.

The present invention also comprises the fragment of separating and the oligonucleotides derived from SEQ ID NO:2-3 and 6 nucleotide sequence. The present invention also comprises by these fragments and the coded polypeptide of oligonucleotides.

Because known genetic code has degeneracy, the more than one codon same amino acid of encoding wherein can be arranged, and the dna sequence dna that therefore is different from sequence shown in SEQ ID NO:2-3 and 6 still codified has the furcella polypeptide of the amino acid sequence of SEQ ID NO:7. This type of modification D NA sequence can produce by silent mutation (for example, occuring) in the pcr amplification process, perhaps can be the product that native sequences is carried out autotelic mutagenesis.

Therefore, the invention provides the dna sequence dna of separation of equal value, its coding furcella polypeptide, the described dna sequence dna that is selected from: (a) comprise the nucleic acid molecules of SEQ ID NO:2-3 and 6; (b) can be under high stringency and the DNA of SEQ ID NO:3 or 6 hybridization; (c) comprise the nucleic acid molecules of the fragment of SEQ ID NO:2-3 and 6; (d) because of the genetic codon degeneracy by (a), (b) or DNA (c) nucleic acid molecules that produce and coding furcella polypeptide and furcella polypeptide fragment. The furcella polypeptide coded by this class nucleic acid equivalent sequence also is within the scope of the present invention.

Include but not limited to furcella polypeptide fragment and the furcella polypeptide that above-mentioned N-glycosylation site, protease Processing position or the conservative amino acid that comprises deactivation replaces by the example with the furcella polypeptide of the dna encoding of SEQ ID NO:3 or 6 equivalences.

Can adopt the method preparation of knowing to contain the recombinant expression carrier of the nucleotide sequence of coding furcella polypeptide. Expression vector comprises and operably is connected in the suitable Spike dna sequence dna of regulating nucleotide sequence of transcribing or translate that for example those are derived from the adjusting nucleotide sequence of mammal, microorganism, virus or insect genes. The example of regulating sequence comprises the suitable sequence of the initial sum termination that transcripting promoter, operon or enhancer, mRNA ribosome bind site and control are transcribed and translated. When regulating sequence and Spike dna sequence dna when function is associated, then nucleotide sequence is " operably connection ". Therefore, if the promoter nucleotide sequence is controlled transcribing of Spike DNA sequence, then the promoter nucleotide sequence is operably to be connected in the Spike dna sequence dna. Replication origin is given the replication capacity in required host cell usually, and the Select gene for the identification of transformant can be mixed expression vector.

In addition, also coding usually can not mixed expression vector with the sequence of the natural relevant suitable signal peptide of furcella polypeptide.

The expression vector that is used for prokaryotic host cell generally includes one or more alternative phenotypic markers gene. Alternative phenotypic markers gene for example is the gene of a kind of coding protein of giving antibiotic resistance or the gene that a kind of coding provides the protein of autotrophy demand. The example that can be used for the expression vector of prokaryotic host cell comprises that those are from the expression vector of commercial plasmid. Commercial carrier comprises that those are the specially designed carrier of marking protein, comprises pMAL-p2 and pMAL-c2 carrier, and they are used for expressing the protein (New England Biolabs, Beverly, MA, USA) that is blended in maltose-binding protein.

The instantiation of plasmid that comprises the SARS CoV Spike gene of optimization comprises following plasmid:

PPCR-Script-040078 was preserved in C.N.C.M. on June 8th, 2004, and preserving number is I-3221;

PcDNA-Spike-HKUPRC-040086 was preserved in C.N.C.M. on June 8th, 2004, and preserving number is I-3222;

PcSFV-HKUPRC-040091 was preserved in C.N.C.M. on June 8th, 2004, and preserving number is I-3223.

The promoter sequence of prokaryotic host cell expression vector of being usually used in recombinating comprises beta-lactamase (penicillase), Lac operon system (Chang et al., Nature 275:615,1978; With Goeddel et al., Nature 281:544,1979), tryptophan (trp) promoter systems (Goeddel et al., Nucl.Acids Res.8:4057,1980; And EP-A-36776), tac promoter (Cold Spring Harbor Laboratory, p. 412,1982 for Maniatis, Molecular Cloning:A Laboratory Manual).

The suitable host cell that is used for expression furcella polypeptide comprises prokaryotic, yeast or higher eucaryotic cells. The suitable clone and the expression vector that are used for bacterium, fungi, yeast and mammalian cell host can be referring to for example Pouwels et al.Cloning Vectors:A Laboratory Manual, Elsevier, New York, (1985). Also can adopt cell free translation system, use the RNA derived from DNA construct described herein to produce the furcella polypeptide.

Be appreciated that the present invention is intended to comprise form separation or purifying of above-mentioned protein, no matter it uses technology described herein or adopts additive method and obtain. Of the present invention one preferred embodiment in, the furcella polypeptide is substantially free of tissue and tissue ingredient of human body tissue ingredient, nucleic acid, irrelevant protein and lipid and external microorganism for example bacterium and virus. Be appreciated that the present invention also comprises basically having described same biological characteristics and immunogenic equivalent protein. Therefore, the present invention is intended to comprise the various serotype variants of protein of the present invention.

According to the purposes of furcella polypeptide of the present invention, can carry out mark to it. The example of suitable label is radioactively labelled substance, enzyme labeling thing, fluorescent marker, chemiluminescent labels and chromophore. The method that is used for mark protein and sugar albumen of the present invention does not have substantial different with the method for the extensive employing of marked immunoglobulin institute. If adopt the antibody for the mark of antigen of the present invention, perhaps use the AIG of anti-antibody for described antigen as indirect labels, then need not again mark antigen of the present invention.

Obtain after the furcella polypeptide of the present invention, available its generation has reactive polyclone and monoclonal antibody with it. Therefore, can come immune animal with protein of the present invention or polypeptide by technology known in the art. This type of technology comprises inoculation, but also can comprise other method of application. Use the protein of capacity or polypeptide in order in the animal reservoir, produce immune response. Can use any host that can produce for the antibody of antigen of the present invention. Animal by immunity and through enough normal time so that after it begins to produce antibody for described antigen, can collect polyclonal antibody. Conventional method comprises from the animal blood sampling, and the autoblood separation of serum. Contain serum for the antibody of described antigen and can be used as antiserum for antigen. Perhaps, can collect antibody from serum. The optimization technique for the polyclonal antibody of antigen of collecting purifying from serum is affinity purification.

Also can prepare the monoclonal antibody for antigen of the present invention. Generation comprises with antigen-immunized animal with the step that described antigen has a kind of method of reactive monoclonal antibody; Spleen from the host is collected the cell that produces antibody; The cell that produces antibody is merged to form hybridoma with the myeloma cell with hypoxanthine-guanine phosphoribosyltransferase defective; Select at least a hybridoma by growth in the culture medium that comprises hypoxanthine, aminopterin and thymidine; Identify at least a generation for the hybridoma of the antibody of described antigen, the hybridoma that cultivation is identified is to produce the antibody of recyclable amount; And collect the antibody that the hybridoma by described cultivation produces.

Polyclone or monoclonal antibody can serve many purposes, and one of them is the corresponding protein of neutralization. They also can be used for the viral antigen in the detection of biological goods, perhaps are used for the corresponding protein of purifying, glycoprotein or its mixture, for example use affinity column to carry out.

The furcella polypeptide can be used as antigen and comes the antibody for SARS CoV in the characterization of biological material, and is used for determining the concentration of antibody in the described material. Therefore, described antigen can be used for the virus in qualitative or quantitatively definite material. Materials comprises tissue and human body cell and biological fluid, and people's body fluid for example comprises people's serum. When the immunoassay of the antibody that is used for determining whether existing anti-SARS CoV as reagent or its concentration, antigen of the present invention provides a kind of simple, quick, responsive and special determination method.

More specifically, antigen of the present invention can be used for detecting SARS CoV by immunoassay, these immunoassays are known can be used for measuring or quantitative liquid in body fluid components (humoral components in fluids). Like this, can directly observe or by second the reaction for example the precipitation or aggegation measure antigen antibody interaction. In addition, also can use immuno-electrophoresis. For example, can use agarose electrophoresis then to make up with this classics of antiserum reaction, and immune labeled (Western trace or the Western blotting) of dielectrophoresis, rocket electrophoresis and polyacrylamide gel electrophoresis. Can use other immunoassays of antigen of the present invention to include but not limited to radioimmunoassay, competition immunoprecipitation assay, enzyme immunoassay and immunofluorescence assay. Be appreciated that and also can use turbidimetry, colorimetric method and nephelometric analysis technology. Be preferably based on the immunoassay of western blotting technique.

One of immunoreagent (antigen of the present invention or for the antibody of the present invention of described antigen) can be immobilized onto a kind of carrier surface when carrying out immunoassay and still keep the immunoreactivity of this reagent. The opposing party's immunoreagent can be also still to keep its immunoreactivity behind cold or the mark. These technology are particularly suitable for enzyme immunoassay, for example enzyme-linked immunosorbent assay (ELISA) CIEIA method (CIEIA).

If be attached to solid support with antigen of the present invention or for the antibody of antigen, described solid support is glass or plastic material normally. Preferably plastic material is shaped to the form of template, cast, pearl or dish. The form of suitable plastic material is polystyrene and polyvinyl chloride. If immunoreagent is difficult for being incorporated into solid support, then a kind of carrier material can be set between reagent and solid support. The example of suitable carrier material is for example bovine serum albumin(BSA) of protein, perhaps for example glutaraldehyde (gluteraldehyde) or urea of chemical reagent. Can adopt routine techniques to be coated with solid support.

The invention provides immunogenicity furcella polypeptide, more specifically for the preparation of the protective polypeptide of the vaccine combination of anti-SARS CoV. Therefore described polypeptide can be applied to as viral vaccine SARS CoV is infected the mammal with neurological susceptibility. Can adopt conventional method of application. For example, can use by oral, respiratory tract or the outer approach of stomach and intestine. When outside adopting stomach and intestine, using, be preferably intracutaneous, subcutaneous and intramuscular routes is used.

The whole bag of tricks of realizing the vaccine adjuvant effect comprises and uses for example aluminium oxide or aluminum phosphate (alum), usually use the solution of 0.05%-0.1%, be dissolved in the phosphate-buffered salt, and the synthetic polymer (Carbopol) of mixed sugar, it uses 0.25% solution. Another kind of suitable adjuvant compound comprises DDA (DDA (dimethyldioctadecyl-ammonium bromide)), and immunomodifier for example lymphokine (such as IFN-γ, IL-1, IL-2 and IL-12) or IFN-γ derivant compound such as poly I:C.

Vaccine composition of the present invention preferably is prepared as injectable form (liquid solution or suspension).But, also can be prepared as the solid phase form that in liquid, is mixed with solution or suspension before being adapted at injecting.

In addition, if desired, vaccine composition can contain a spot of supplementation material, and for example wetting agent or emulsifying agent, pH buffer reagent or adjuvant are to strengthen the effectiveness of vaccine.

The method of application of vaccine composition of the present invention and its dosage formulation forms fit, and its amount can produce result of treatment and immunogenicity.The amount of using depends on individuality to be treated, comprises that for example the immunity system of individuality is induced the ability that produces immunne response.

The dosage of vaccine depends on route of administration, and changes along with patient's age to be inoculated, and on less degree, changes along with the stature size for the treatment of the inoculator.

The main purpose that infects the intravital immunne response of Mammals of SARS CoV is the free SARS CoV of deactivation and removes because of infecting the cell that SARS CoV may discharge infectious virus.The immunne response major responsibility deactivation SARS CoV virus of B cell aspect.The main mode that realizes this purpose is that infectivity is neutralized.Another important mechanisms of destroying the cell that infects SARS CoV is provided by cytotoxic T lymphocyte (CTL), unites the viral Spike antigen of expression on the CTL recognizing cells surface with I class histocompatibility antigen.The furcella polypeptide that comes from spike protein processing in the CTL recognizing cells, described spike protein is produced by the cell that infects, or by the phagocytic cell internalization.Therefore, the present invention can be used for stimulating the B cell response of anti-furcella polypeptide, and the immunity that is mediated by CTL behind the virus infection.CTL replys the rehabilitation of infecting at former SARS CoV of mediation and quicken aspect such as rehabilitation in course of infection subsequently and plays a significant role.

Can strengthen furcella polypeptide of the present invention and vaccine are induced the neutralizing antibody of protectiveness level in host ability by carrying out emulsifying effect with adjuvant, mix liposome, being coupled to suitable carrier or the combination by these technology.For example, furcella polypeptide of the present invention can be used with conventional adjuvant, and these adjuvants are aluminum phosphate and alumina gel for example, presents in an amount at least sufficient to strengthen in host body fluid or cell-mediated immune responses.Similarly, the furcella polypeptide can be incorporated into lipid film or mix lipid film to form liposome.Can use the nonthermal source lipid that does not contain nucleic acid and other no related substances for this reason.

Immunization scheme depends on multiple factor, and for example the host is to the susceptibility of infection and host's age.Can use the vaccine of the present invention of single dose to the host, perhaps carry out initial immunization earlier, use several dosage at interval with certain hour then.After initial inoculation, as required dosage is subsequently used as booster shot.

The amount that is applied to host's spike protein of the present invention, polypeptide and vaccine will be enough to prevent or suppress duplicate in the infection of SARS CoV or the body.Under any circumstance, the amount of using all should be enough to protect the host to avoid occurring tangible immunosuppression at least, infects even can't prevent SARS CoV fully.Can produce immunogenic response by use spike protein of the present invention or glycoprotein to the host, the amount of using is about 10 to about 500 microgram antigen/kg body weight, is preferably about 50 to about 100 microgram antigen/kg body weight.Protein of the present invention and vaccine can be used with the physiology acceptable carrier.For example, can use thinner, for example water or salts solution.

Another aspect of the present invention provides the method for RNA and/or dna immunization inoculation.This method also comprises the arbitrary combination of using nucleic acid, described protein and the polypeptide itself of coding furcella polypeptide to individuality, adds or the no carrier added(NCA) molecule.In embodiment, described individuality is an animal, and Mammals preferably.More preferably, Mammals is selected from people, mouse, rat, rabbit, sheep, dog, cat, ox, pig and horse.In particularly preferred embodiments, Mammals is the people.

Methods of treatment comprises uses the immunogenic composition that comprises the furcella polypeptide, and the composition of using the nucleic acid of coding furcella polypeptide also can.Those skilled in the art know nucleic acid vaccine and nucleic acid vaccine technology and based on technology conception, application and the validity of protein and polypeptide.Technology based on nucleic acid can be applied directly to tissue and cell with the nucleic acid of coding furcella polypeptide that expose or capsulation, and need not produce coded protein before using.This technology is based on these nucleic acid and can and expresses with generation immunogenicity determinant by the cellular uptake of receptor organism, and receptor's immunity system can be replied its generation.Typically, expressed antigen is showed in picked-up and has expressed the cell surface of described nucleic acid, but the present invention also comprises coded antigen presentation and exports in the recycle system of receptor's individuality.This type of nucleic acid vaccine technology includes but not limited to carry naked DNA and RNA and carries coding furcella polypeptide expression carrier.Although this technology is called " vaccine ", it also is applicable to the immunogenic composition that does not produce protective response on an equal basis.This type of does not induce the composition of protectiveness and method to belong to scope of the present invention yet.

The nucleic acid of furcella polypeptide and carrier molecule are carried as naked nucleic acid and are belonged to scope of the present invention although will encode, and the present invention also comprises nucleic acid is carried as the part of bigger or more complicated composition.These delivery systems comprise virus, virus-like particle or the bacterium of the nucleic acid that contains coding furcella polypeptide.Equally, nucleic acid of the present invention and carrier molecule and permeability cell compound (cell permeabilizing compounds) also belong to scope of the present invention as the formed complex body of liposome.Other compounds that are used for nucleic acid vaccine for example molecular vehicle (EP 696,191, Samain etal.) and delivery system be that those skilled in the art are known, for example referring to WO 93 06223 and WO 9011092, U.S.5,580,859 and U.S.5,589,466 (patents of Vical) are incorporated these documents into this paper by reference, and above-mentioned these all need not carry out too much experiment can be prepared and use.

Behind single or double injection spike protein, thisly can induce neutrality and protection antibody dependent immune response based on proteinic SARS vaccine.Virus vaccines (that is, plasmid, MVA, Adeno) or inactivated whole virus vaccines based on proteinic vaccine and vector expression are compared the advantage that shows suitable security.

In order further to realize purpose of the present invention and consistent, provide a kind of test kit that is used to diagnose SARS CoV infection with purpose of the present invention.In one embodiment, described test kit contains antibody of the present invention, and described antibody can be in conjunction with SARS CoV furcella polypeptide.In another embodiment, described test kit contains polypeptide of the present invention, and described polypeptide can detect whether there is the antibody that can combine with the furcella polypeptide.In another embodiment, described test kit contains nucleic acid molecule of the present invention, and described nucleic acid molecule can be hybridized with viral RNA or similar dna sequence dna that prompting exists SARS CoV to infect.Spendable various diagnostic techniques includes but not limited to: (1) be used to identify can by or the Southern trace method of the cell DNA of not being limited property enzymic digestion; (2) be used to identify that extraction is from the Northern of the RNA of cell trace; (3) dot hybridization technology promptly need not to separate on sepharose in advance, directly sample is filtered by Ad Hoc film, for example nitrocellulose filter or nylon membrane; (4) based on the immunoassay of western blotting technique; (5) ELISA (enzyme-linked immunosorbent assay); (6) FACS; (7) indirect immunofluorescence assay method; Or (8) immunoprecipitation assay.The suitable material that is used for the dot hybridization technology can derive from body fluid, includes but not limited to, the supernatant liquor of serum and blood plasma, culturing cell or lysis are also by the film of centrifugal removal cell and the endochylema extract after the nucleus.

Further set forth the present invention below in conjunction with embodiment.

Embodiment 1

Obtain to come the patient's of self-infection SARS CoV furcella gene (Spike gene), be called Spike-Pasteur (SEQ ID NO:1).Spike-Pasteur is cloned into the pcDNA carrier for expression of eukaryon and is transfected into the 293T cell.Transfection the cell of pcDNA-Spike-Pasteur do not express the Spike-Pasteur polypeptide, but because all do not observe the spike protein of detection level by FACS or Western trace.

Express Spike-Pasteur subsequently in the SFV virus expression carrier, described expression vector can be expressed in the bhk cell of transfection effectively.But, the productive rate of SFV virion is lower, and this is because there are 2 Spe I sites in the furcella gene.Spe I is generally used for plasmid is carried out linearizing at the end of SFV encoding sequence.Owing to can not use Spe I, therefore used Sph I that PSFV-Spike-Pasteur is carried out linearizing, this has produced＞extra 3 ' RNA sequence of the vector rna of 2000 bases.Come self-infection the representative expression level of cell of SFV-Spike-Pasteur very weak.

In order to strengthen the expression of Spike-Pasteur, two Spe I sites in the Spike-Pasteur sequence are eliminated, produced Spike-Pasteur-modif (SEQ ID NO:2).Spike-Pasteur-modif makes it possible to Spe I pSFV be carried out the linearizing of standard, and finds that it has improved 100 times with SFV particulate productive rate maximum.From the common SFV titre that produces of the RNA transfection of the linearizing pSFV-Spike-Pasteur of Sph I is 2 * 10 ⁷IP/ml.From the common SFV titre that produces of the RNA transfection of the linearizing pSFV-Spike-Pasteur-modif of Spe I is 1-2 * 10 ⁹IP/ml.

Embodiment 2

Further the Spike-Pasteur sequence is carried out bioinformatic analysis.The cDNA of Spike-Pasteur contains many cis actings site, and negative impact can be caused to expression in these sites.To express in order further strengthening, in Spike-Pasteur, to have eliminated 32 in 33 negative cis acting signals being identified, and added the extra signal that stimulated gene is expressed, produced Spike-HKU-PRC (SEQ ID NO:3) thus.Spike-HKU-PRC is cloned into pSC, pcDNA and pSFV carrier.

As shown in table 1,19 RNA unstable motifs that are rich in AU of Spike-Pasteur all are eliminated to produce Spike-HKU-PRC.11 and inner poly (A) and tumor-necrosis factor glycoproteins and secondary tract in 12 donor splicing sites of inferring and the acceptor site have been removed in addition.

Table 1. is compared the negative cis acting signal among the Spike-Pasteur with the Spike-HKU-PRC that optimizes.

	Spike-Pasteur	Spike-HKU-PRC
			Be rich in the RNA unstable motif of AU	19	0
Tumor-necrosis factor glycoproteins and secondary tract	1	0
			Donor splicing site and acceptor site	12	1
Inner poly (A) site	1	0

Add extra expression enhancement sequences on the Spike-HKU-PRC to and comprise the Kozak consensus sequence that is incorporated into initial ATG upstream strengthening the initial of translation, and added 2 STOP codons to guarantee effective termination.In order to increase the transformation period of mRNA, the GC content of Spike-HKU-PRC is increased to 49% by 38%, avoid occurring the zone of GC content high (＞80%) or extremely low (＜30%) simultaneously.In addition, the codon of use is partial to Chinese hamster to improve translation efficiency.The codon that table 2 is depicted as Chinese hamster uses, and the frequency representation of each codon is quantity/1,000 codon.

Following sequence is avoided appearing among the Spike-HKU-PRC: inner TATA box, chi site and ribosome entry site(RES); Be rich in AT or be rich in the tract of GC; Tumor-necrosis factor glycoproteins and RNA secondary structure; With donor splicing site and acceptor site and montage branching-point (splice branch points).

The codon of table 2. Chinese hamster uses, and sees http://www.kazusa.or.ip/codon/).Frequency representation is quantity/1,000 codon.

UUU UUC UUA UUG	19.3 22.0 6.1 14.1	UCU UCC UCA UCG	16.1 16.5 10.1 3.5	UAU UAC UAA UAG	12.8 16.3 0.6 0.6	UGU UGC UGA UGG	8.9 10.3 1.1 13.3
								CUU CUC CUA CUG	12.9 18.1 7.5 38.6	CCU CCC CCA CCG	17.3 17.4 15.5 4.3	CAU CAC CAA CAG	10.1 13.0 10.2 33.8	CGU CGC CGA CGG	5.8 9.2 7.1 10.2
AUU AUC AUA AUG	17.4 25.0 6.8 22.8	ACU ACC ACA ACG	14.2 20.5 15.7 4.3	AAU AAC AAA AAG	17.3 21.1 24.3 38.4	AGU AGC AGA AGG	11.6 16.3 9.9 10.2
								GUU GUC GUA GUG	11.6 15.9 7.9 30.2	GCU GCC GCA GCG	22.7 25.8 16.5 4.8	GAU GAC GAA GAG	24.7 27.9 27.6 40.9	GGU GGC GGA GGG	13.2 21.8 16.3 13.7

Embodiment 3

Come the spike protein of comparison pcDNA-Spike-Pasteur and pcDNA-Spike-HKU-PRC to express by using the calcium phosphate method that plasmid transfection is gone into the 293T cell.Transfection do not observe spike protein in the cell of pcDNA-Spike-Pasteur.On the contrary, in transfection detect high-caliber spike protein (Fig. 1) in the 293T cell of pcDNA-Spike-HKU-PRC.The migration of spike protein is consistent with the result who obtained in the past with the oligomerization mode, points out this plasmid can express SARS CoV protein total length, native conformation.These results confirm that theatrical improvement takes place the codon optimized expression of results that makes of Spike encoding sequence.

Embodiment 4

With the peptide-labeled spike protein of C end Flag, as shown in Figure 2.In the formerly described SFV carrier system (referring to Staropoli et al., Lozach et al. and Chanel et al. all incorporate this paper at this by reference), spike protein is expressed as full length protein, comprises C end and membrane spaning domain and C end Flag marker.

Perhaps from transfection SFV-Spike-RNA cell or infected the SFV particulate cell preparation spike protein of coding SFV-Spike-RNA.Preparation SFV expression vector RNA

Analyze the correct folding and expection characteristic of spike protein by a series of biochemistry and immunocytochemical assay method.This protein obtains high mannose EndoH susceptibility N-glycan (Fig. 3) entering endoplasmic reticulum (ER) generation glycosylation.Express (Fig. 4), solubility ACE2 receptors bind (Fig. 5) and in the Western trace, discerned and confirm by ER quality control exit, plasma membrane by facs analysis (Fig. 6-7) by the SARS patients serum, the folding of spike protein is correct, is its native conformation.

Embodiment 5

For carrying out the RNA immunization, according to disclosed standard program in-vitro transcription RNA.

In bhk cell, produce spike protein, and use anti-FLAG M2 antibody by the affine purifying that under natural condition, carries out of immunity.Use the spike protein of Flag peptide elution of bound M2 under natural condition.Remove peptide and residual washing agent by dialysis.

Intramuscular injection SFV Spike RNA immune mouse is subsequently at the 14th day and the 35th day intraperitoneal (IP) injection spike protein.Obtained serum at the 34th, 42 and 55 day from mice immunized, described serum shows existence reorganization Spike specific antibody in Western trace (Fig. 6) and FACS (Fig. 7), and carries out the immunofluorescence detection and find that there is SARS CoV specific antibody (Fig. 8) in described serum on the FRHK4 cell that has infected SARS CoV.These data show, the spike protein of expressing in the SFV carrier can its native conformation and successfully carry out immune purifying, and the protein of this purifying can be induced the anti-SARS antibody that produces high titre mouse.

Embodiment 6

Following reagent and method can be used for implementing the present invention.

Produce SARS CoV Spike subunit vaccine

1/ preparation SFV expression vector RNA

Annotate: spike protein can produce from, for example, transfection the cell of SFV-Spike-RNA or the cell that infects with the SFV particle of coding SFV-Spike-RNA.Describe a kind of Electroporaton method in detail at this.

Preparation 1.2 * 10 ⁷Cell/ml suspension is used for carrying out under the aseptic condition electroporation (Electroporation)

1. preparation serum free medium, the also filtration/degerming of mixed following composition:

i.Hepes 5％10mL

Ii. tryptose-phosphate broth (Tryptose-phosphate broth) 50mL

Iii. penicillin 100U/mL, Streptomycin sulphate 100 μ g/mL:5mL

iv.GMEM QSP 500mL

2. (GMEM, trypsinase, PBS do not contain Ca to pre-thermal agent ²⁺Or Mg ²⁺) to 37 ℃;

3. gently absorb former substratum, do not contact wall;

4. with 10ml PBS flushing cell once, abandon washing lotion;

5. add 3ml trypsinase and with placement 4-5 minute in cover (hood) of flat culturing bottle;

6. add fresh perfect medium of 17ml (GMEM 5%FCS) and suspension cell, be transferred in the 50ml pipe;

7. cell counting; Get and be equivalent to 10 ⁷Cell and at 1500rpm centrifugal 5 minutes;

8. cell is resuspended in 1ml PBS and (obtains 10 ⁷Cell/ml);

9. suspension is placed on ice.

2/ transfection SFV expression vector RNA

Should all carry out electroporation to the control cells of sample and untransfected.

1. prepare 2 75ml culturing bottles, one of them contains 20ml GMEM, suitably mark;

2. use aseptic P1000 filter suction nozzle to shift 800 μ l cell suspensions and (be suspended from no Ca ²⁺Or Mg ²⁺PBS in) to the pipe that contains RNA, inhale with pipettor and to make a call to twice;

3. mixture is transferred to rapidly in the electroporation pipe (Electroporator cuvette) that has been placed in the electroporation chamber;

4. electroporation apparatus is set in 830 volts, 25 μ Fd, and with impedance setting in infinity, apply 2 pulses, the 2-3 delay of second (annotate: for reaching suitable time constant, keeping electroporation chamber when applying pulse is crested) is arranged between each pulse;

5. note the electroporation time (should within 0.4ms);

With cell transfer to the 75ml culturing bottle that contains 20ml GMEM, gently inhale and beat with re-suspended cell;

7. on the cell of untransfected, carry out same operation;

With cell at 37 ℃, 5%CO ₂(about 16 hours) are incubated overnight under the condition.

3/ lysing cell also prepares albumen

Preparation is used for the protein cleavage thing of Western trace and immune purifying

Lysis buffer

Triton X-100 1％

Tris-HCI，pH 7.5 20mM

NaCl 150mM

EDTA 1mM

PMSF 50mg/ml

1 * PBS does not contain Ca ²⁺, Mg ²⁺

The protein sample damping fluid does not contain DTT

DTT，1M，-20℃

Cell scraper

Prepare fresh 20ml lysis buffer

1. remove the substratum in the culturing bottle;

2. with 10ml 1 * PBS washed cell (1 bottle);

3. add 500 μ l lysates, utilize cell scraper in culturing bottle, to take out cell;

4. carefully cell pyrolysis liquid is transferred in the eppendorf pipe of 1.5ml;

5. take out residual cell as much as possible with 300 extra μ l lysis buffers;

6. pipe was placed on ice 15 minutes;

With pipe 4 ℃ with 13, centrifugal 15 minutes of 000rpm is to remove nuclear material;

8. supernatant liquor is transferred to the new pipe that places on ice;

9. the lysate extract is positioned on ice.

4/ in cell pyrolysis liquid immunoaffinity purification S-Flag protein

Triton X 100 lysis buffers (Triton X 100 1%, Tris HCl pH7.5 20mM, NaCl 150mM, EGTA 1mM, PMSF 50 μ g/ml)

1. using the wealthy mouth of 200 μ l to move the liquid head places 1.5mleppendorf to be used for each lysis sample the anti-flag M2 sepharose 4B of 100 μ l;

2. with the pearl of each pipe of 1ml lysis buffer balance, wash 3 times (pearl got rid of to the bottom in centrifugal 15 seconds at full speed, gently remove 90% PBS, attention is avoided pearl is picked up);

3. preserve the cell lysate of 50 μ l, standby;

4. fully mixed (soft rotation) pearl-cleavage mass mixture, the pearl after will washing thus with remaining cell lysate 4 ℃ of incubations 4 hours;

5. pearl is got rid of to the bottom, and remove supernatant liquor from each sample hose;

6. with 0.5ml 1X lavation buffer solution washing pearl 3 times (get rid of to the bottom, and add new lavation buffer solution);

7. pearl is got rid of to the bottom, and remove most supernatant liquors, make that the resid vol in each sample hose is about 100 μ l;

8. each pearl sample with 20 μ l is dispensed in the new 1.5ml eppendorf pipe, is used for the Western trace and detects;

9. remaining pearl sample hose is deposited in-20 ℃, be used for wash-out after preparing.

Elute protein in immunoprecipitate

1. method is with reference to the FLAGIPT-1 working instructions;

2. with 3XFLAG peptide wash-out;

3. by being that the 1X lavation buffer solution that the 3X FLAG peptide of 5 μ g/ μ l is added into 100 μ l prepares 3X FLAG peptide working fluid with 3 μ l concentration;

4. the 3X FLAG work elutriant with 100 μ l adds on the resin;

With mixture 4 ℃ of incubations 1 hour, soft rotation;

6. with resin centrifugal 10 seconds at 13000rpm;

7. reservation supernatant liquor, and step 4-6 repeated 3 times again.

Concentrate and protein purification with the Amicon filter device

1. behind the incubation supernatant liquor of each culturing bottle is collected in the 50mL pipe;

2. centrifugal 5 minutes of 2000rpm is to remove cell precipitation;

3. the 15mL supernatant liquor is transferred to (to contain the PMSF of the 100mM of 57 μ L, be dissolved in iPrOH) in another 50mL pipe and chooses wantonly

4. place on ice;

5. the 20ml sample is added to Amicon Ultra-15 filter device;

6. the filter device of building is placed in the centrifugal rotor, volume markings towards the top, is carried out counterweight with similar device;

7. in horizontal rotor (swinging bucket rotor) centrifugal 20 minutes with maximum 4000xg;

8. reclaim spissated solute (500 μ l) by drawing sample from filter device.

Embodiment 7

After measured, candidate vaccine preparation, i.e. tripolymer S albumen (TriSpike, the protein identical), purity＞90% with described Spike-HKU-PRC.To be used for that the purifying TriSpike sample that mouse and hamster carry out immunization research is carried out sex change at SDS/DTT damping fluid (50mM DDT) and become monomer so that trimer protein matter is dissociated fully.Separate with 4-12%SDS-PAGE, gel is carried out silver dye (Current Protocols in Immunology Chapter 8,9.1-9.10) all proteins to be contained in the show sample.Figure 16 demonstration is merely able to detect monomer S albumen, has purity＞90% with its compound glycosylation and high mannose form.

Embodiment 8

The animal that adds the Alum adjuvant immunity with TriSpike has produced the enhanced serum IgG and has replied.In the past to studies show that of replying from the mucous membrane of the reorganization HagB of porphyromonas gingivalis (Porphyromonas gingivalis) and whole body, compare with the HagB immunity that does not add adjuvant, in the Balb/c mouse, induced higher serum IgG to reply with the HagB+Alum immunity and replied (Vaccine with mucous membrane IgA, 2003,21,4459-4471).The TriSpike candidate vaccine is analyzed determining that it whether not only can blood serum induced IgG, and can be induced the mucous membrane IgA that SARS CoV is had neutralising capacity.TriSpike preparation among the PBS and the TriSpike preparation in the Alum adjuvant have been compared in the ability of inducing aspect the SARS CoV specific serum IgG.By the intraperitoneal approach two groups of mouse are carried out the proteic mouse of 20 μ g TriSpike that 3 dosage are accepted in the representative of immunity: A group separately, and the mouse of the 20 μ g TriSpike that mixed 1mg Alum adjuvant in advance of 3 dosage is accepted in the representative of B group.The Western engram analysis shows, carries out mice immunized with independent use TriSpike and compares, and carries out mice immunized with TriSpike+Alum and has produced stronger antibody response (Figure 17).TriSpike+Alum group also demonstrate higher in and titre (Figure 17).The TriSpike+Alum adjuvant has induced intensive neutrality and persistent serum IgG to reply.

Embodiment 9

The TriSpike+Alum adjuvant has induced enhanced mucous membrane IgG and IgA to reply.The upper respiratory tract and lower respiratory tract human and experimental infection animal can detect SARS CoV.Except respiratory tract, in the intestinal tissue of death, also can detect SARS CoV (AJG, 2005,100,169-176).Induce the ability that produces SARS CoV specific IgG and IgA antibody in order to study the TriSpike candidate vaccine in mucosal sites, the contriver collected from TriSpike+/-the Alum adjuvant carries out the ight soil and the nose irrigating solution sample of mice immunized by the intraperitoneal approach.Prepare as previously mentioned faecal samples (PNAS, 2004,101,13584-13589).In brief, collect the excrement piece (100mg) in the appointed date.40 ℃ of soft rotations 30 minutes of the PBS that contains 0.02%Na-azide that add 0.5ml, and centrifugal (13,000rpm), be prepared into stool extract thus.Usually, can be prepared into the supernatant liquor of 0.2ml from a night soil management piece suspension.Western engram analysis (Figure 18) shows, only exists mucous membrane IgG and IgA to reply in the faecal samples with the TriSpike+Alum mice immunized, does not then have in separately with the TriSpike mice immunized.Similarly, only have from Ig contained in the faecal samples with the TriSpike+Alum mice immunized and in little neutralization analysis, demonstrate neutralization activity with anti-SARS CoV.Detected IgG and IgA in from the faecal samples with the TriSpike+Alum mice immunized, this explanation has been set up the first line defense mechanism that anti-SARS CoV infects with the animal of TriSpike immunity in gastrointestinal system.

(Chapter 19,11.15-16) described preparation nose irrigating solution sample as Current Protocols in Immunology.In brief, collect nose irrigating solution sample in the appointed date.Mouse is implemented anesthesia for originally ketamine/xylazine solution of mouse and mice immunized intraperitoneal injection two volumes.Open the thoracic cavity, insert the 25G syringe needle, 0.5ml PBS/ Trypsin inhibitor,Trasylol is injected in the tracheae chamber of blocking size portion.Can collect the nose scavenging solution sample of about 0.5ml from each mouse.

The nose irrigating solution sample of collecting (n=3) is carried out the Western engram analysis find, in nose irrigating solution sample, have mucous membrane IgG, then do not have with the TriSpike mice immunized separately with the TriSpike+Alum mice immunized.But, all do not detect IgA and reply in TriSpike+Alum mice immunized or independent nose irrigating solution sample with the TriSpike mice immunized, this may be by due to the antigen route of administration.According to little neutralization analysis result, reply external from the mucous membrane IgG of nose sample and not produce the provide protection that any anti-SARS CoV infects.

Sequence table

<110>INSTITUT PASTEUR

HONG KONG PASTEUR RESEARCH CENTRE LIMITED

＜120〉nucleic acid, polypeptide, expression method and the immunogenic composition relevant with sars cov spike protein

＜130〉B6401AA Abandon D/CAL

<140>PCT/EP 2005/006512

<141>2005-06-03

<150>10/860,641

<151>2004-06-04

<150>60/578,348

<151>2004-06-10

<160>7

<170>PatentIn Ver.3.3

<210>1

<211>3795

<212>DNA

<213>SARS Coronavirus

<400>1

atgtttattt tcttattatt tcttactctc actagtggta gtgaccttga ccggtgcacc 60

acttttgatg atgttcaagc tcctaattac actcaacata cttcatctat gaggggggtt 120

tactatcctg atgaaatttt tagatcagac actctttatt taactcagga tttatttctt 180

ccattttatt ctaatgttac agggtttcat actattaatc atacgtttgg caaccctgtc 240

atacctttta aggatggtat ttattttgct gccacagaga aatcaaatgt tgtccgtggt 300

tgggtttttg gttctaccat gaacaacaag tcacagtcgg tgattattat taacaattct 360

actaatgttg ttatacgagc atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420

tctaaaccca tgggtacaca gacacatact atgatattcg ataatgcatt taattgcact 480

ttcgagtaca tatctgatgc cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540

cacttacgag agtttgtgtt taaaaataaa gatgggtttc tctatgttta taagggctat 600

caacctatag atgtagttcg tgatctacct tctggtttta acactttgaa acctattttt 660

aagttgcctc ttggtattaa cattacaaat tttagagcca ttcttacagc cttttcacct 720

gctcaagaca tttggggcac gtcagctgca gcctattttg ttggctattt aaagccaact 780

acatttatgc tcaagtatga tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840

aatccacttg ctgaactcaa atgctctgtt aagagctttg agattgacaa aggaatttac 900

cagacctcta atttcagggt tgttccctca ggagatgttg tgagattccc taatattaca 960

aacttgtgtc cttttggaga ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020

gagagaaaaa aaatttctaa ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080

ttttcaacct ttaagtgcta tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140

aatgtctatg cagattcttt tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200

caaactggtg ttattgctga ttataattat aaattgccag atgatttcat gggttgtgtc 1260

cttgcttgga atactaggaa cattgatgct acttcaactg gtaattataa ttataaatat 1320

aggtatctta gacatggcaa gcttaggccc tttgagagag acatatctaa tgtgcctttc 1380

tcccctgatg gcaaaccttg caccccacct gctcttaatt gttattggcc attaaatgat 1440

tatggttttt acaccactac tggcattggc taccaacctt acagagttgt agtactttct 1500

tttgaacttt taaatgcacc ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560

aagaaccagt gtgtcaattt taattttaat ggactcactg gtactggtgt gttaactcct 1620

tcttcaaaga gatttcaacc atttcaacaa tttggccgtg atgtctctga tttcactgat 1680

tccgttcgag atcctaaaac atctgaaata ttagacattt caccttgctc ttttgggggt 1740

gtaagtgtaa ttacacctgg aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800

gttaactgca ctgatgtttc tacagcaatc catgcagatc aactcacacc agcttggcgc 1860

atatattcta ctggaaacaa tgtattccag actcaagcag gctgtcttat aggagctgag 1920

catgtcgaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac 1980

catacagttt ctttattacg tagtactagc caaaaatcta ttgtggctta tactatgtct 2040

ttaggtgctg atagttcaat tgcttactct aataacacca ttgctatacc tactaacttt 2100

tcaattagca ttactacaga agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160

aatatgtaca tctgcggaga ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220

ttttgcacac aactaaatcg tgcactctca ggtattgctg ctgaacagga tcgcaacaca 2280

cgtgaagtgt tcgctcaagt caaacaaatg tacaaaaccc caactttgaa atattttggt 2340

ggttttaatt tttcacaaat attacctgac cctctaaagc caactaagag gtcttttatt 2400

gaggacttgc tctttaataa ggtgacactc gctgatgctg gcttcatgaa gcaatatggc 2460

gaatgcctag gtgatattaa tgctagagat ctcatttgtg cgcagaagtt caatgggctt 2520

acagtgttgc cacctctgct cactgatgat atgattgctg cctacactgc tgctctagtt 2580

agtggtactg ccactgctgg atggacattt ggtgctggcg ctgctcttca aatacctttt 2640

gctatgcaaa tggcatatag gttcaatggc attggagtta cccaaaatgt tctctatgag 2700

aaccaaaaac aaatcgccaa ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760

acaacaacat caactgcatt gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820

ttaaacacac ttgttaaaca acttagctct aattttggtg caatttcaag tgtgctaaat 2880

gatatccttt cgcgacttga taaagtcgag gcggaggtac aaattgacag gctaattaca 2940

ggcagacttc aaagccttca aacctatgta acacaacaac taatcagggc tgctgaaatc 3000

agggcttctg ctaatcttgc tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060

agagttgact tttgtggaaa gggctaccac cttatgtcct tcccacaagc agccccgcat 3120

ggtgttgtct tcctacatgt cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180

ccagcaattt gtcatgaagg caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240

ggcacttctt ggtttattac acagaggaac ttcttttctc cacaaataat tactacagac 3300

aatacatttg tctcaggaaa ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360

gatcctctgc aacctgagct tgactcattc aaagaagagc tggacaagta cttcaaaaat 3420

catacatcac cagatgttga tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480

attcaaaaag aaattgaccg cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540

gaccttcaag aattgggaaa atatgagcaa tatattaaat ggccttggta tgtttggctc 3600

ggcttcattg ctggactaat tgccatcgtc atggttacaa tcttgctttg ttgcatgact 3660

agttgttgca gttgcctcaa gggtgcatgc tcttgtggtt cttgctgcaa gtttgatgag 3720

gatgacgctg agccagttct caagggtgtc aaattacatt acacagacta caaggatgac 3780

gatgacaata agtaa 3795

<210>2

<211>3795

<212>DNA

<213>SARS Coronavirus

<400>2

atgtttattt tcttattatt tcttactctc acgagtggta gtgaccttga ccggtgcacc 60

acttttgatg atgttcaagc tcctaattac actcaacata cttcatctat gaggggggtt 120

tactatcctg atgaaatttt tagatcagac actctttatt taactcagga tttatttctt 180

ccattttatt ctaatgttac agggtttcat actattaatc atacgtttgg caaccctgtc 240

atacctttta aggatggtat ttattttgct gccacagaga aatcaaatgt tgtccgtggt 300

tgggtttttg gttctaccat gaacaacaag tcacagtcgg tgattattat taacaattct 360

actaatgttg ttatacgagc atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420

tctaaaccca tgggtacaca gacacatact atgatattcg ataatgcatt taattgcact 480

ttcgagtaca tatctgatgc cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540

cacttacgag agtttgtgtt taaaaataaa gatgggtttc tctatgttta taagggctat 600

caacctatag atgtagttcg tgatctacct tctggtttta acactttgaa acctattttt 660

aagttgcctc ttggtattaa cattacaaat tttagagcca ttcttacagc cttttcacct 720

gctcaagaca tttggggcac gtcagctgca gcctattttg ttggctattt aaagccaact 780

acatttatgc tcaagtatga tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840

aatccacttg ctgaactcaa atgctctgtt aagagctttg agattgacaa aggaatttac 900

cagacctcta atttcagggt tgttccctca ggagatgttg tgagattccc taatattaca 960

aacttgtgtc cttttggaga ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020

gagagaaaaa aaatttctaa ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080

ttttcaacct ttaagtgcta tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140

aatgtctatg cagattcttt tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200

caaactggtg ttattgctga ttataattat aaattgccag atgatttcat gggttgtgtc 1260

cttgcttgga atactaggaa cattgatgct acttcaactg gtaattataa ttataaatat 1320

aggtatctta gacatggcaa gcttaggccc tttgagagag acatatctaa tgtgcctttc 1380

tcccctgatg gcaaaccttg caccccacct gctcttaatt gttattggcc attaaatgat 1440

tatggttttt acaccactac tggcattggc taccaacctt acagagttgt agtactttct 1500

tttgaacttt taaatgcacc ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560

aagaaccagt gtgtcaattt taattttaat ggactcactg gtactggtgt gttaactcct 1620

tcttcaaaga gatttcaacc atttcaacaa tttggccgtg atgtctctga tttcactgat 1680

tccgttcgag atcctaaaac atctgaaata ttagacattt caccttgctc ttttgggggt 1740

gtaagtgtaa ttacacctgg aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800

gttaactgca ctgatgtttc tacagcaatc catgcagatc aactcacacc agcttggcgc 1860

atatattcta ctggaaacaa tgtattccag actcaagcag gctgtcttat aggagctgag 1920

catgtcgaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac 1980

catacagttt ctttattacg tagtactagc caaaaatcta ttgtggctta tactatgtct 2040

ttaggtgctg atagttcaat tgcttactct aataacacca ttgctatacc tactaacttt 2100

tcaattagca ttactacaga agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160

aatatgtaca tctgcggaga ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220

ttttgcacac aactaaatcg tgcactctca ggtattgctg ctgaacagga tcgcaacaca 2280

cgtgaagtgt tcgctcaagt caaacaaatg tacaaaaccc caactttgaa atattttggt 2340

ggttttaatt tttcacaaat attacctgac cctctaaagc caactaagag gtcttttatt 2400

gaggacttgc tctttaataa ggtgacactc gctgatgctg gcttcatgaa gcaatatggc 2460

gaatgcctag gtgatattaa tgctagagat ctcatttgtg cgcagaagtt caatgggctt 2520

acagtgttgc cacctctgct cactgatgat atgattgctg cctacactgc tgctctagtt 2580

agtggtactg ccactgctgg atggacattt ggtgctggcg ctgctcttca aatacctttt 2640

gctatgcaaa tggcatatag gttcaatggc attggagtta cccaaaatgt tctctatgag 2700

aaccaaaaac aaatcgccaa ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760

acaacaacat caactgcatt gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820

ttaaacacac ttgttaaaca acttagctct aattttggtg caatttcaag tgtgctaaat 2880

gatatccttt cgcgacttga taaagtcgag gcggaggtac aaattgacag gctaattaca 2940

ggcagacttc aaagccttca aacctatgta acacaacaac taatcagggc tgctgaaatc 3000

agggcttctg ctaatcttgc tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060

agagttgact tttgtggaaa gggctaccac cttatgtcct tcccacaagc agccccgcat 3120

ggtgttgtct tcctacatgt cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180

ccagcaattt gtcatgaagg caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240

ggcacttctt ggtttattac acagaggaac ttcttttctc cacaaataat tactacagac 3300

aatacatttg tctcaggaaa ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360

gatcctctgc aacctgagct tgactcattc aaagaagagc tggacaagta cttcaaaaat 3420

catacatcac cagatgttga tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480

attcaaaaag aaattgaccg cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540

gaccttcaag aattgggaaa atatgagcaa tatattaaat ggccttggta tgtttggctc 3600

ggcttcattg ctggactaat tgccatcgtc atggttacaa tcttgctttg ttgcatgacg 3660

agttgttgca gttgcctcaa gggtgcatgc tcttgtggtt cttgctgcaa gtttgatgag 3720

gatgacgctg agccagttct caagggtgtc aaattacatt acacagacta caaggatgac 3780

gatgacaata agtaa 3795

<210>3

<211>3897

<212>DNA

<213>SARS Coronavirus

<220>

<221>CDS

<222>(44)..(3844)

<400>3

ctatagggcg aattgggtac cgctagcgga tccgcgcgcc acc atg ttt att ttc 55

Met Phe Ile Phe

1

ctg ctg ttt ctg act ctg acc agc ggc agt gac ctg gac cgg tgc acc 103

Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu Asp Arg Cys Thr

5 10 15 20

act ttt gat gat gtg cag gct cct aat tac act cag cat act tcc tct 151

Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln His Thr Ser Ser

25 30 35

atg agg ggc gtg tac tat cct gat gaa att ttt aga tcc gac act ctg 199

Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg Ser Asp Thr Leu

40 45 50

tat ctg act cag gat ctg ttt ctg cca ttc tat tct aat gtg aca ggc 247

Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser Asn Val Thr Gly

55 60 65

ttt cat act att aat cat acc ttt ggc aac cct gtg atc cct ttt aag 295

Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val Ile Pro Phe Lys

70 75 80

gat ggc atc tat ttt gct gcc aca gag aag tcc aat gtg gtg cgg gga 343

Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn Val Val Arg Gly

85 90 95 100

tgg gtg ttc ggc tct acc atg aac aac aag tcc cag tcc gtg att att 391

Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln Ser Val Ile Ile

105 110 115

att aac aat tct act aat gtg gtg atc cga gcc tgt aac ttt gaa ctg 439

Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys Asn Phe Glu Leu

120 125 130

tgt gac aac cca ttc ttt gct gtg tct aag ccc atg ggc aca cag aca 487

Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met Gly Thr Gln Thr

135 140 145

cat act atg atc ttc gat aat gcc ttt aat tgc act ttc gag tac atc 535

His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr Phe Glu Tyr Ile

150 155 160

tct gat gcc ttt tcc ctg gat gtg tcc gaa aag tcc ggc aac ttt aag 583

Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys

165 170 175 180

cac ctg cga gag ttt gtg ttt aag aat aag gat ggc ttt ctg tat gtg 631

His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val

185 190 195

tat aag ggc tat cag cct atc gac gtg gtg cgc gat ctg cct tct ggc 679

Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly

200 205 210

ttt aac act ctg aag cct att ttt aag ctg cct ctg ggc att aac att 727

Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile

215 220 225

aca aat ttt cgg gcc att ctg aca gcc ttt agc cct gct cag gac att 775

Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro Ala Gln Asp Ile

230 235 240

tgg ggc acc tct gct gcc gcc tat ttt gtg ggc tat ctg aag cca act 823

Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr

245 250 255 260

acc ttt atg ctg aag tat gat gaa aat ggc aca atc aca gat gct gtg 871

Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val

265 270 275

gat tgt tct cag aat cca ctg gct gaa ctg aag tgc tct gtg aag agc 919

Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser

280 285 290

ttt gag att gac aag gga atc tac cag acc tct aat ttc cgc gtg gtg 967

Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val

295 300 305

ccc tct gga gat gtg gtg aga ttc cct aat att aca aac ctg tgt cct 1015

Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro

310 315 320

ttt gga gaa gtg ttt aat gct act aag ttc cct tct gtg tat gcc tgg 1063

Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala Trp

325 330 335 340

gag aga aag aag att tct aat tgt gtg gct gat tac tct gtg ctg tac 1111

Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr

345 350 355

aac tcc aca ttt ttt agc acc ttt aag tgc tat ggc gtg tct gcc act 1159

Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala Thr

360 365 370

aag ctg aat gat ctg tgc ttc tcc aat gtg tat gcc gat tct ttt gtg 1207

Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val

375 380 385

gtg aag gga gat gat gtg aga cag atc gcc cca gga cag act ggc gtg 1255

Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Val

390 395 400

att gct gat tac aat tat aag ctg cca gat gat ttc atg ggc tgt gtg 1303

Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met Gly Cys Val

405 410 415 420

ctg gct tgg aat act agg aac att gat gct act tcc act ggc aat tat 1351

Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr

425 430 435

aat tac aag tat cgg tat ctg aga cat ggc aag ctg agg ccc ttt gag 1399

Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe Glu

440 445 450

aga gac atc tct aac gtg cct ttc agc cct gat ggc aag cct tgc acc 1447

Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr

455 460 465

cca cct gct ctg aat tgt tat tgg cca ctg aat gat tat ggc ttt tac 1495

Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe Tyr

470 475 480

acc act act ggc att ggc tac cag cct tac aga gtg gtg gtg ctg tct 1543

Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser

485 490 495 500

ttt gaa ctg ctg aat gcc cct gcc aca gtg tgt gga cca aag ctg tcc 1591

Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro Lys Leu Ser

505 510 515

act gac ctg att aag aac cag tgt gtg aac ttt aac ttt aat gga ctg 1639

Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn Phe Asn Gly Leu

520 525 530

act ggc act ggc gtg ctg act cct tct agc aag aga ttt cag cca ttt 1687

Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg Phe Gln Pro Phe

535 540 545

cag cag ttt ggc cgg gat gtg tct gat ttc act gat tcc gtg cga gat 1735

Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp Ser Val Arg Asp

550 555 560

cct aag aca tct gaa atc ctg gac att tcc cct tgc tct ttt ggc ggc 1783

Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys Ser Phe Gly Gly

565 570 575 580

gtg agc gtg att aca cct gga aca aat gct tcc tct gaa gtg gct gtg 1831

Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu Val Ala Val

585 590 595

ctg tat cag gat gtg aac tgc act gat gtg tct aca gcc atc cat gcc 1879

Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr Ala Ile His Ala

600 605 610

gat cag ctg aca cca gct tgg cgc atc tat tct act gga aac aat gtg 1927

Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val

615 620 625

ttc cag act cag gcc ggc tgt ctg atc gga gct gag cat gtg gac act 1975

Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu His Val Asp Thr

630 635 640

tct tat gag tgc gac att cct att gga gct ggc att tgt gct agt tac 2023

Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr

645 650 655 660

cat aca gtg tct ctg ctg cgg agt act agc cag aag tct att gtg gct 2071

His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys Ser Ile Val Ala

665 670 675

tat act atg tct ctg ggc gct gat agt tcc att gct tac tct aat aac 2119

Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala Tyr Ser Asn Asn

680 685 690

acc att gct atc cct act aac ttt tcc att agc att act aca gaa gtg 2167

Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile Thr Thr Glu Val

695 700 705

atg cct gtg tct atg gct aag acc tcc gtg gat tgt aat atg tac atc 2215

Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys Asn Met Tyr Ile

710 715 720

tgc gga gat tct acc gaa tgt gct aat ctg ctg ctg cag tat ggc agc 2263

Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu Gln Tyr Gly Ser

725 730 735 740

ttt tgc aca cag ctg aat cgg gct ctg tct ggc att gct gct gaa cag 2311

Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile Ala Ala Glu Gln

745 750 755

gat cgc aac aca cgg gaa gtg ttc gct caa gtg aag cag atg tat aag 2359

Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys Gln Met Tyr Lys

760 765 770

acc cca act ctg aag tat ttt ggc ggc ttt aat ttt tcc cag atc ctg 2407

Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu

775 780 785

cct gac cct ctg aag ccc act aag cgg tct ttt att gag gac ctg ctg 2455

Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile Glu Asp Leu Leu

790 795 800

ttt aac aaa gtg aca ctg gct gat gct ggc ttt atg aag cag tat ggc 2503

Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly

805 810 815 820

gaa tgc ctg ggc gat att aat gct aga gat ctg att tgt gcc cag aag 2551

Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile Cys Ala Gln Lys

825 830 835

ttc aat ggc ctg aca gtg ctg cct cct ctg ctg act gat gat atg att 2599

Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Asp Met Ile

840 845 850

gct gcc tac act gct gct ctg gtg tct ggc act gcc act gct gga tgg 2647

Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala Thr Ala Gly Trp

855 860 865

aca ttt ggc gct ggc gct gct ctg cag atc cct ttt gct atg cag atg 2695

Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met

870 875 880

gcc tat cgg ttc aat ggc att gga gtg acc cag aat gtg ctg tat gag 2743

Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu

885 890 895 900

aac cag aag cag att gcc aac cag ttt aac aag gcc att agt cag att 2791

Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln Ile

905 910 915

cag gaa tcc ctg aca aca aca tcc act gcc ctg ggc aag ctg cag gac 2839

Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln Asp

920 925 930

gtg gtg aac cag aat gct cag gcc ctg aac aca ctg gtg aag cag ctg 2887

Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu

935 940 945

agc agc aat ttt ggc gcc att tcc agt gtg ctg aat gat atc ctg tcc 2935

Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser

950 955 960

cga ctg gat aaa gtg gag gcc gaa gtg cag att gac agg ctg att aca 2983

Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr

965 970 975 980

ggc aga ctg cag agc ctg cag acc tat gtg aca cag cag ctg atc agg 3031

Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg

985 990 995

gct gct gaa atc agg gct tct gcc aat ctg gct gct act aag atg tct 3079

Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser

1000 1005 1010

gag tgt gtg ctg gga cag tcc aag aga gtg gac ttt tgt gga aag ggc 3127

Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly

1015 1020 1025

tac cac ctg atg tcc ttc cca cag gct gcc cct cat gga gtg gtg ttc 3175

Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe

1030 1035 1040

ctg cat gtg acc tat gtg cca tcc cag gag agg aac ttc acc aca gcc 3223

Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala

1045 1050 1055 1060

cca gcc att tgt cat gaa ggc aag gcc tac ttc cct cgg gaa ggc gtg 3271

Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val

1065 1070 1075

ttc gtg ttt aat ggc act tct tgg ttt att aca cag cgg aac ttc ttt 3319

Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe

1080 1085 1090

agc cca cag atc atc act aca gac aat aca ttt gtg tcc gga aat tgt 3367

Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys

1095 1100 1105

gat gtg gtg att ggc atc att aac aac aca gtg tat gat cct ctg cag 3415

Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln

1110 1115 1120

cct gag ctg gac tcc ttc aag gaa gag ctg gac aag tac ttc aag aat 3463

Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1125 1130 1135 1140

cat aca tcc cca gat gtg gat ctg ggc gac att tcc ggc att aac gct 3511

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala

1145 1150 1155

tct gtg gtg aac att cag aag gaa att gac cgc ctg aat gaa gtg gct 3559

Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala

1160 1165 1170

aag aat ctg aat gaa tcc ctg att gac ctg cag gaa ctg ggc aag tat 3607

Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr

1175 1180 1185

gag cag tat att aag tgg cct tgg tat gtg tgg ctg ggc ttc att gct 3655

Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile Ala

1190 1195 1200

gga ctg att gcc atc gtg atg gtg aca atc ctg ctg tgt tgc atg acc 3703

Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys Met Thr

1205 1210 1215 1220

tcc tgt tgc agt tgc ctg aag ggc gct tgc tct tgt gga tct tgc tgc 3751

Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly Ser Cys Cys

1225 1230 1235

aag ttt gat gag gat gac tct gag cca gtg ctg aag ggc gtg aag ctg 3799

Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu

1240 1245 1250

cat tac aca ggg ccc ggc ggc gac tac aag gac gat gac gac aag 3844

His Tyr Thr Gly Pro Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys

1255 1260 1265

tgatagatcg atgcatggat ccgtttaaac cgagctccag ctttgttccc tta 3897

<210>4

<211>1267

<212>PRT

<213>SARS Coronavirus

<400>4

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr Gly Pro Gly Gly Asp Tyr Lys Asp Asp

1250 1255 1260

Asp Asp Lys

1265

<210>5

<211>1267

<212>PRT

<213>SARS Coronavirus

<400>5

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr Gly Pro Gly Gly Asp Tyr Lys Asp Asp

1250 1255 1260

Asp Asp Lys

1265

<210>6

<211>3765

<212>DNA

<213>SARS Coronavirus

<400>6

atgtttattt tcctgctgtt tctgactctg accagcggca gtgacctgga ccggtgcacc 60

acttttgatg atgtgcaggc tcctaattac actcagcata cttcctctat gaggggcgtg 120

tactatcctg atgaaatttt tagatccgac actctgtatc tgactcagga tctgtttctg 180

ccattctatt ctaatgtgac aggctttcat actattaatc atacctttgg caaccctgtg 240

atccctttta aggatggcat ctattttgct gccacagaga agtccaatgt ggtgcgggga 300

tgggtgttcg gctctaccat gaacaacaag tcccagtccg tgattattat taacaattct 360

actaatgtgg tgatccgagc ctgtaacttt gaactgtgtg acaacccatt ctttgctgtg 420

tctaagccca tgggcacaca gacacatact atgatcttcg ataatgcctt taattgcact 480

ttcgagtaca tctctgatgc cttttccctg gatgtgtccg aaaagtccgg caactttaag 540

cacctgcgag agtttgtgtt taagaataag gatggctttc tgtatgtgta taagggctat 600

cagcctatcg acgtggtgcg cgatctgcct tctggcttta acactctgaa gcctattttt 660

aagctgcctc tgggcattaa cattacaaat tttcgggcca ttctgacagc ctttagccct 720

gctcaggaca tttggggcac ctctgctgcc gcctattttg tgggctatct gaagccaact 780

acctttatgc tgaagtatga tgaaaatggc acaatcacag atgctgtgga ttgttctcag 840

aatccactgg ctgaactgaa gtgctctgtg aagagctttg agattgacaa gggaatctac 900

cagacctcta atttccgcgt ggtgccctct ggagatgtgg tgagattccc taatattaca 960

aacctgtgtc cttttggaga agtgtttaat gctactaagt tcccttctgt gtatgcctgg 1020

gagagaaaga agatttctaa ttgtgtggct gattactctg tgctgtacaa ctccacattt 1080

tttagcacct ttaagtgcta tggcgtgtct gccactaagc tgaatgatct gtgcttctcc 1140

aatgtgtatg ccgattcttt tgtggtgaag ggagatgatg tgagacagat cgccccagga 1200

cagactggcg tgattgctga ttacaattat aagctgccag atgatttcat gggctgtgtg 1260

ctggcttgga atactaggaa cattgatgct acttccactg gcaattataa ttacaagtat 1320

cggtatctga gacatggcaa gctgaggccc tttgagagag acatctctaa cgtgcctttc 1380

agccctgatg gcaagccttg caccccacct gctctgaatt gttattggcc actgaatgat 1440

tatggctttt acaccactac tggcattggc taccagcctt acagagtggt ggtgctgtct 1500

tttgaactgc tgaatgcccc tgccacagtg tgtggaccaa agctgtccac tgacctgatt 1560

aagaaccagt gtgtgaactt taactttaat ggactgactg gcactggcgt gctgactcct 1620

tctagcaaga gatttcagcc atttcagcag tttggccggg atgtgtctga tttcactgat 1680

tccgtgcgag atcctaagac atctgaaatc ctggacattt ccccttgctc ttttggcggc 1740

gtgagcgtga ttacacctgg aacaaatgct tcctctgaag tggctgtgct gtatcaggat 1800

gtgaactgca ctgatgtgtc tacagccatc catgccgatc agctgacacc agcttggcgc 1860

atctattcta ctggaaacaa tgtgttccag actcaggccg gctgtctgat cggagctgag 1920

catgtggaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac 1980

catacagtgt ctctgctgcg gagtactagc cagaagtcta ttgtggctta tactatgtct 2040

ctgggcgctg atagttccat tgcttactct aataacacca ttgctatccc tactaacttt 2100

tccattagca ttactacaga agtgatgcct gtgtctatgg ctaagacctc cgtggattgt 2160

aatatgtaca tctgcggaga ttctaccgaa tgtgctaatc tgctgctgca gtatggcagc 2220

ttttgcacac agctgaatcg ggctctgtct ggcattgctg ctgaacagga tcgcaacaca 2280

cgggaagtgt tcgctcaagt gaagcagatg tataagaccc caactctgaa gtattttggc 2340

ggctttaatt tttcccagat cctgcctgac cctctgaagc ccactaagcg gtcttttatt 2400

gaggacctgc tgtttaacaa agtgacactg gctgatgctg gctttatgaa gcagtatggc 2460

gaatgcctgg gcgatattaa tgctagagat ctgatttgtg cccagaagtt caatggcctg 2520

acagtgctgc ctcctctgct gactgatgat atgattgctg cctacactgc tgctctggtg 2580

tctggcactg ccactgctgg atggacattt ggcgctggcg ctgctctgca gatccctttt 2640

gctatgcaga tggcctatcg gttcaatggc attggagtga cccagaatgt gctgtatgag 2700

aaccagaagc agattgccaa ccagtttaac aaggccatta gtcagattca ggaatccctg 2760

acaacaacat ccactgccct gggcaagctg caggacgtgg tgaaccagaa tgctcaggcc 2820

ctgaacacac tggtgaagca gctgagcagc aattttggcg ccatttccag tgtgctgaat 2880

gatatcctgt cccgactgga taaagtggag gccgaagtgc agattgacag gctgattaca 2940

ggcagactgc agagcctgca gacctatgtg acacagcagc tgatcagggc tgctgaaatc 3000

agggcttctg ccaatctggc tgctactaag atgtctgagt gtgtgctggg acagtccaag 3060

agagtggact tttgtggaaa gggctaccac ctgatgtcct tcccacaggc tgcccctcat 3120

ggagtggtgt tcctgcatgt gacctatgtg ccatcccagg agaggaactt caccacagcc 3180

ccagccattt gtcatgaagg caaggcctac ttccctcggg aaggcgtgtt cgtgtttaat 3240

ggcacttctt ggtttattac acagcggaac ttctttagcc cacagatcat cactacagac 3300

aatacatttg tgtccggaaa ttgtgatgtg gtgattggca tcattaacaa cacagtgtat 3360

gatcctctgc agcctgagct ggactccttc aaggaagagc tggacaagta cttcaagaat 3420

catacatccc cagatgtgga tctgggcgac atttccggca ttaacgcttc tgtggtgaac 3480

attcagaagg aaattgaccg cctgaatgaa gtggctaaga atctgaatga atccctgatt 3540

gacctgcagg aactgggcaa gtatgagcag tatattaagt ggccttggta tgtgtggctg 3600

ggcttcattg ctggactgat tgccatcgtg atggtgacaa tcctgctgtg ttgcatgacc 3660

tcctgttgca gttgcctgaa gggcgcttgc tcttgtggat cttgctgcaa gtttgatgag 3720

gatgactctg agccagtgct gaagggcgtg aagctgcatt acaca 3765

<210>7

<211>1255

<212>PRT

<213>SARS Coronavirus

<400>7

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr

1250 1255

Claims (57)

1. the nucleic acid molecule of a purifying, it comprises SEQ ID NO:2 (Spike-Pasteur-modif), SEQ ID NO:3 (Spike-HKU-PRC) or SEQ ID NO:6.

2. the nucleic acid molecule of a purifying, its a kind of aminoacid sequence of encoding, described aminoacid sequence comprises the sequence of SEQ ID NO:4 or SEQ ID NO:7, and the nucleic acid molecule of wherein said purifying is compared the expression that demonstrates the enhanced spike protein with SEQ ID NO:1.

3. the nucleic acid molecule of a purifying, its arbitrary chain with the double-stranded DNA of the sex change of the nucleotide sequence that comprises SEQ ID NO:3 or SEQ ID NO:6 is hybridized under high stringency.

4. the nucleic acid molecule of the purifying of claim 3, the nucleic acid molecule of wherein said purifying is compared the expression that demonstrates the enhanced spike protein with SEQID NO:1.

5. the nucleic acid molecule of the purifying of claim 4, it comprises the displacement of at least a negative cis acting signal, and the peptide sequence of wherein coded described spike protein does not change.

6. the nucleic acid molecule of the purifying of claim 5, wherein said negative cis acting signal comprise at least a in following one group:

(a) be rich in the RNA unstable motif of AU;

(b) tumor-necrosis factor glycoproteins;

(c) secondary tract (secondary stretch);

(d) donor splicing site and acceptor site; With

(e) inner poly (A) site.

7. the nucleic acid molecule of the purifying of claim 6, the nucleic acid molecule of wherein said purifying also comprises at least a extra expression enhancement sequences.

8. the nucleic acid molecule of the purifying of claim 7, wherein said extra expression enhancement sequences comprise at least a with in next group:

(a) Kozak consensus sequence; With

(b) extra STOP codon.

9. the nucleic acid molecule of the purifying of claim 4, wherein the use of codon is optimized according to the preference of Chinese hamster (Cricetulus griseus).

10. the nucleic acid molecule of the purifying of claim 4, the part of the described spike protein of coding of the nucleic acid molecule of wherein said purifying is compared with SEQ ID NO:1, its GC percentage composition increase about at least 10%.

11. the nucleic acid molecule of the purifying of claim 7, the displacement of wherein said at least a negative cis acting signal and wherein said at least a extra expression enhancement sequences do not comprise:

(a) inner TATA box, chi site and ribosome entry site(RES);

(b) be rich in AT or be rich in the tract of GC;

(c) tumor-necrosis factor glycoproteins and RNA secondary structure; With

(d) donor splicing site and acceptor site.

12. a recombinant vectors, its guidance is selected from the expression of the nucleic acid molecule of the nucleic acid molecule of each purifying in the claim 1 to 3.

13. the polypeptide of a purifying, it comprises SEQ ID NO:4, SEQ ID NO:5 or SEQID NO:7.

14. the polypeptide of a purifying, it is by the nucleic acid molecule encoding that is selected from the nucleic acid molecule of each purifying in the claim 1 to 3.

15. the polypeptide of the purifying of claim 14, the polypeptide of wherein said purifying comprise high mannose EndoH susceptibility N-glycan.

16. antibody purified, the polypeptide of described antibodies claim 14.

17. the antibody purified of claim 16, wherein said antibody is monoclonal antibody.

18. the antibody purified of claim 16, wherein said antibody comprises neutralizing antibody.

19. with the carrier transfection of claim 12 or the host cell of transduction.

20. the host cell of claim 19, wherein said host cell are selected from 293T cell, bhk cell and FRHK4 cell.

21. strengthen the method for expressing SARS CoV furcella polypeptide by nucleic acid molecule, comprise the quantity that reduces the negative cis acting signal in the described nucleic acid molecule, wherein the reduction of the quantity of negative cis acting signal does not change the sequence of described SARS CoV furcella polypeptide.

22. the method for claim 21, wherein said negative cis acting signal comprise at least a in following one group: the RNA unstable motif that is rich in AU; Tumor-necrosis factor glycoproteins; The secondary tract; Donor splicing site and acceptor site; With inner poly (A) site.

23. the method for claim 22, wherein said method also comprise the sequence of introducing extra signal and not changing described SARS CoV furcella polypeptide.

24. the method for claim 23, wherein said extra signal comprise at least a in Kozak consensus sequence and the extra STOP codon.

25. the method for claim 24 also comprises the use according to the preference optimizing codon of Chinese hamster.

26. the method for claim 24 also comprises the GC content increase about at least 10% in the coding region of furcella Nucleotide.

27. an isolating immunocomplex, it comprises the antibody of SARS CoV furcella polypeptide and the described polypeptide of specific recognition.

28. an isolating immunocomplex, it comprises the antibody of SARS CoV furcella polypeptide and the described polypeptide of specific recognition, and wherein said antibody is to produce at the polypeptide of the purifying of claim 14.

29. comprising providing, a method that detects the SARS virus infection, wherein said method comprise the doubtful composition that has infected the biologic material of SARS virus, and the existence of measuring the furcella polypeptide.

30. the method for claim 29 is wherein measured described furcella polypeptide by electrophoresis or by the use and the immunoassay of the antibody of described furcella polypeptide generation immunological response.

31. one kind is detected the method that SARS virus infects, wherein said method comprises providing and comprises the doubtful composition that has infected the biologic material of SARS virus, and the existence of measuring the furcella polypeptide, wherein said antibody is that the polypeptide at the purifying of claim 14 produces.

32. be used to detect the in-vitro diagnosis method that whether exists in conjunction with comprising the antigenic antibody of SARS CoV furcella polypeptide, wherein said method comprises described antigen is contacted with a kind of biological fluid, the time of contact and condition are enough to make that the antibody in described antigen and the biological fluid forms immune complex, and the formation that detects described mixture.

33. the method for claim 32, it also comprises the formation of measuring described immune complex.

34. the method for claim 33 wherein detects the formation of immune complex by the immunoassay based on western blotting technique, ELISA, indirect immunofluorescence assay method or immunoprecipitation assay.

35. be used to detect the in-vitro diagnosis method of antigenic antibody that whether exists in conjunction with the polypeptide of the purifying that comprises claim 14, wherein said method comprises described antigen is contacted with a kind of biological fluid, the time of contact and condition are enough to make that the antibody in described antigen and the biological fluid forms immune complex, and the formation that detects described mixture.

36. be used to detect the diagnostic kit that whether exists in conjunction with the antibody of SARS CoV furcella polypeptide or its mixture, described test kit comprises the antigen of the mixture that comprises SARS CoV furcella polypeptide or SARS CoV furcella polypeptide, and being used to detect the reagent that forms immunocomplex between described antigen and the antibody, the amount of wherein said reagent is enough to carry out described detection.

37. be used to detect the diagnostic kit that whether exists in conjunction with the antibody of SARS CoV furcella polypeptide or its mixture, described test kit comprises the antigen of the polypeptide of the purifying that comprises claim 14, and being used to detect the reagent that forms immunocomplex between described antigen and the antibody, the amount of wherein said reagent is enough to carry out described detection.

38. an immunogenic composition, it comprises at least a SARS CoV furcella polypeptide and medicinal acceptable carrier, and the amount of described SARS CoV furcella polypeptide is enough to induction of immunity originality or protective response in vivo.

39. the immunogenic composition of claim 38, wherein said composition comprise at least a SARS CoV furcella polypeptide of dosis neutralisata.

40. the immunogenic composition of claim 38 also comprises the Alum adjuvant.

41. an immunogenic composition, it comprises the polypeptide and the medicinal acceptable carrier of the purifying of claim 14, and the amount of described polypeptide is enough to induction of immunity originality or protective response in vivo.

42. handle host's method with the immunogenic composition of claim 41, comprise to the host and use described immunogenic composition that the amount of the immunogenic composition of being used is enough to induction of immunity originality or protective response in vivo.

43. the method for claim 42 is wherein used described immunogenic composition by the intraperitoneal approach.

44. the method for claim 42, wherein said immunogenic composition also comprises the Alum adjuvant.

45. the method for claim 42, wherein said immunogenic composition is used according to a dosage scheme, and described dosage comprises twice or repeatedly use the SARS CoV furcella peptide of 2-20 μ g.

46. the vaccine composition of an anti-SARS CoV, it comprises the polypeptide of claim 14.

47. the methods of vaccination of an anti-SARS CoV, it comprises the vaccine composition of using claim 46 to the animal that needs are arranged.

48. the method for claim 47, wherein said methods of vaccination are induced enhanced mucous membrane IgA and IgG antibody.

49. the method for claim 48, wherein said vaccine composition also comprises the Alum adjuvant.

50. the method for claim 49, wherein said vaccine composition is used according to a dosage scheme, and described dosage comprises twice or repeatedly use SARS CoV furcella peptide and the Alum adjuvant of 2-20 μ g.

51. whether a detection exists the method for SARS CoV, it comprises:

(1) sample of the doubtful SARS of containing CoV viral genetic is contacted with at least a nucleotide probe and

(2) hybridization between the viral genetic in described nucleotide probe of detection and the sample, wherein said nucleotide probe is complementary to the full length sequence of the purification of nucleic acid of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:6.

52. a plasmid, it is preserved in C.N.C.M, and preserving number is I-3221, I-3222 or I-3223.

53. plasmid-encoded SARS CoV furcella polypeptide by claim 52.

54. the segmental polynucleotide of encoding SARS CoV furcella polypeptide, it is compared with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:6 has at least a sudden change.

55. the nucleotide sequence of claim 54 or the fragment of polynucleotide, it comprises at least 10 successive Nucleotide and maximum 150 successive Nucleotide.

56. a polynucleotide compositions, it comprises the nucleotide sequence of claim 54 or 55 at least.

57. each polypeptide or polynucleotide among claim 1-11,13-15 and the 53-55, the t cell response that it can induce anti-SARS to infect.

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