CN1768074B

CN1768074B - A diagnostic assay for the human virus causing severe acute respiratory syndrome (SARS)

Info

Publication number: CN1768074B
Application number: CN200480007678.7A
Authority: CN
Inventors: 陈国雄; 管轶; J·M·黎国思; J·S·M·裴伟士; 潘烈文; 袁国勇
Original assignee: Versitech Ltd
Current assignee: Versitech Ltd
Priority date: 2003-03-24
Filing date: 2004-03-24
Publication date: 2010-04-28
Anticipated expiration: 2024-03-24
Also published as: CN1768140B; CN100543132C; CN1768140A; CN1768074A; CN101186920A; CN1761745A

Abstract

The present invention relates to a diagnostic assay for the virus causing Severe Acute Respiratory Syndrome (SARS) in humans (''hSARS virus''). In particular, the invention relates to a real-time quantitative PCR assay for the detection of hSARS virus using reverse transcription and polymerase chain reaction. Specifically, the quantitative assay is a TaqMan(R) assay using the primers and probes constructed based on the genome of the hSARS virus. The invention further relates to a diagnostic kit that comprises nucleic acid molecules for the detection of the hSARS virus.

Description

Diagnostic test to the Human virus that causes severe acute respiratory syndrome (SARS)

The application requires the right of priority of following application: the U.S. Provisional Application 60/457 that on March 24th, 2003 submitted to, 031, the U.S. Provisional Application 60/457 that on March 26th, 2003 submitted to, 730, the U.S. Provisional Application 60/459 that on April 2nd, 2003 submitted to, 931, the U.S. Provisional Application 60/460 that on April 3rd, 2003 submitted to, 357, the U.S. Provisional Application 60/461 that on April 8th, 2003 submitted to, 265, the U.S. Provisional Application 60/462 that on April 14th, 2003 submitted to, 805, the U.S. Provisional Application 60/464 that on April 23rd, 2003 submitted to, 886, the U.S. Provisional Application 60/468 that on May 5th, 2003 submitted to, the U.S. Provisional Application 60/471 that on May 16th, 139 and 2003 submitted to, 200, they separately by reference integral body be attached to herein.

The application comprises a long sequence table, and this sequence table replaces the papery text of printing to submit in the lump by in triplicate CD-R, its by reference integral body be attached to herein.Described CD-R is labeled as " CRF ", " Copy 1 " and " Copy 2 " respectively in imprinting on March 22 in 2004, and each only contains an identical 1.58MB file (V9661078.APP).

1. invention field

The present invention relates to diagnostic test to the virus (" hSARS virus ") that in the mankind, causes severe acute respiratory syndrome (SARS).Specifically, the present invention relates to use reverse transcription and polymerase chain reaction (RT-PCR), measure hSARS virus, it is natural or the quantitative test method of artificial variant, analogue or derivative.In particular, described quantitative test is

Test.The invention still further relates to the diagnostic kit that comprises nucleic acid molecule, be used to detect hSARS virus.

2. background

Recently, atypical pneumonia has been broken out in the Guangdong Province of China's Mainland.Between year March in November, 2002 to 2003,792 routine cases have been reported, wherein dead (WHO.SevereAcute Respiratory Syndrome (SARS) the Weekly Epidemiol Rec.2003 of 31 examples; 78:86).For tackling this crisis, Hong Kong Hospital Authority has strengthened the supervision to serious atypical pneumonia patient.In this fact-finding process, confirmed that a plurality of health care workers suffer from this disease.In addition, a plurality of pneumonia cases appear in the philtrum of the close contact of sick therewith the infected.Although adopted at the known typical antibiotic therapy method of the bacterial pathogens relevant with atypical pneumonia usually, this sick severity and development are still unusual.Inventor of the present invention is one of group that participates in these patients of investigation.In these patients, all negative to all qualification test results of common virus and bacterium.This disease is by the initial name (" SARS ") of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome).After inventor of the present invention isolates hSARS virus from SARS patient, just understood this sick pathogeny factor.The invention provides quick and specificity real time quantitative PCR method disclosed herein.The present invention can be used for clinical and the scientific research purposes.

3. summary of the invention

The present invention relates to the purposes of sequence information in diagnostic method of isolating hSARS virus.In a preferred embodiment, isolating hSARS virus is deposited in Genbank, and NCBI, its searching number are AY278491 (SEQ ID NO:15), and it is attached to herein by reference.Isolating hSARS virus is deposited in Chinese typical culture center (CCTCC) on April 2nd, 2003, is given searching number CCTCC-V200303, and described in following the 7th joint, it is attached to herein by reference.

In a specific embodiments, the invention provides hSARS virus, it is natural or the diagnostic test method of artificial variant, analogue or derivative.Specifically, the present invention relates to use reverse transcription and polymerase chain reaction (RT-PCR), measure the quantitative test method of hSARS viral nucleic acid molecule.In particular, described quantitative test is Test.The nucleic acid molecule that is fit to the hSARS nucleic acid hybridization also is provided among the present invention, for example include but not limited to PCR primer, reversed transcriptive enzyme primer, be used for that Southern analyzes or other nucleic acid hybridization analysis to detect the probe of hSARS nucleic acid.Described hSARS nucleic acid comprises or is made up of nucleotide sequence, and described nucleotide sequence is SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its complement, analogue, derivative, fragment or part.In a preferred embodiment, described primer comprises SEQ ID NO:2471 and/or 2472 nucleotide sequences.In a preferred embodiment, described primer comprises SEQ ID NO:2474 and/or 2475 nucleotide sequences.In a most preferred embodiment, described nucleic acid molecule comprises SEQ IDNO:2473 nucleotide sequence or its part, uses the nucleic acid molecule that comprises SEQ IDNO:2471 and/or 2472 nucleotide sequences to can be used for detecting hSARS virus as primer in the RT-PCR test.In another most preferred embodiment, described nucleic acid molecule comprises SEQ ID NO:2476 nucleotide sequence or its part, uses the nucleic acid molecule that comprises SEQ ID NO:2474 and/or 2475 nucleotide sequences to can be used for detecting hSARS virus as primer in the RT-PCR test.In another most preferred embodiment, described test is

Quantitative test.

In one embodiment, the invention provides and detect hSARS virus or its natural or artificial variant, analogue, the existence of derivative or the method for expression in biomaterial, described biomaterial for example is cell, blood, serum, blood plasma, saliva, urine, excrement, phlegm, nasopharynx aspirate etc.The rising of hSARS virus activity or expression relative comparison sample or reduction can be measured by the reagent that makes the biomaterial contact can directly or indirectly detect existence of hSARS virus or expression in the sample.In a specific embodiments, detection reagent is a nucleic acid molecule of the present invention.In another embodiment, detecting nucleic acid molecule is immobilized on the dna microarray chip.

In a specific embodiments, the invention provides diagnostic kit, wherein comprise the nucleic acid molecule that is suitable for detecting hSARS virus or its natural or artificial variant, analogue, derivative.In a specific embodiments, described nucleic acid molecule has SEQ ID NO:2471 and/or 2472 nucleotide sequences.In specific embodiments, described nucleic acid molecule has SEQ IDNO:2473 nucleotide sequence.In another embodiment, described nucleic acid molecule has SEQ ID NO:2474 and/or 2475 nucleotide sequences.In specific embodiments, described nucleic acid molecule has SEQ ID NO:2476 nucleotide sequence.

In one aspect, the present invention relates to the purposes of isolating hSARS virus in diagnostic method.In a specific embodiments, the invention provides the method that in biomaterial, detects hSARS virus mRNA of the present invention or geneome RNA, described biomaterial for example is cell, blood, serum, blood plasma, saliva, urine, excrement, phlegm, nasopharynx aspirate etc.The rising of hSARS virus mRNA or geneome RNA relative comparison sample or reduction can be measured by the reagent that makes the biomaterial contact can directly or indirectly detect hSARS virus mRNA or geneome RNA in the sample.In a specific embodiments, detection reagent is a nucleic acid molecule of the present invention.In another embodiment, detecting nucleic acid molecule is immobilized on the dna microarray chip.

On the other hand, the present invention relates to the purposes of isolating hSARS virus in diagnostic method, for example in biological sample, detect the antibody of immunologic opsonin in conjunction with hSARS virus.In a specific embodiments, described detection reagent is a hSARS virus, the virus of preserving number CCTCC-V200303 for example, or have the virus of SEQ ID NO:15 genomic nucleic acid sequence, or the polypeptide of SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleic acid sequence encodings.

In yet another aspect, the invention provides antibody or its Fab, its immunologic opsonin is in conjunction with SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequence coded polypeptide of the present invention, be included under the stringent condition and SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474, the polypeptide of the present invention of the nucleic acid encoding of the nucleotide sequence of 2475 or 2476 nucleotide sequence hybridizations, and/or has a bioactive any hSARS epi-position of one or more polypeptide of the present invention.This antibody includes but not limited to polyclonal antibody, monoclonal antibody, bi-specific antibody, multi-specificity antibody, people's antibody, humanized antibody, chimeric antibody, single-chain antibody, Fab fragment, F (ab ') ₂Fv, the intracellular antibody that fragment, disulfide linkage connect and contain the fragment of specificity in conjunction with VL or the VH territory and even the complementary determining region (CDR) of polypeptide of the present invention.

The invention still further relates to the method for discriminating hSARS virus or its natural or artificial variant, analogue, derivative infection object.In a specific embodiments, described method comprises from the biological sample from object and obtains total RNA; The total RNA of reverse transcription is to obtain cDNA; Making cDNA carry out PCR with the primer that derives from the hSARS viral nucleotide sequences with a cover measures.

The invention further relates to the diagnostic kit that comprises primer and nucleic acid probe, to detect the mRNA or the geneome RNA of hSARS virus.

3.1 definition

Term used herein " variant " refers to the natural hereditary variant of hSARS virus or the hSARS viral variants of reorganization preparation, compares with the hSARS virus of CCTCC-V200303, comprises one or more sudden changes in their each comfortable its genomes.Term " variant " also can refer to the natural variant of particular peptide or the particular peptide or the proteic variant of reorganization preparation, and wherein one or more amino-acid residues are modified by amino-acid substitution, insertion or disappearance.

Term used herein " analogue " refers to have second kind of organic or inorganic molecule with first kind of organic or inorganic molecular mimicry or identical function and its structure and first kind of organic or inorganic molecular mimicry for non-albumen analogue.

Term used herein " derivative " refers to second kind of organic or inorganic molecule forming based on the structure of first kind of organic or inorganic molecule for non-protein derivative.The derivative of organic molecule for example includes but not limited to by adding or removing the molecule that hydroxyl, methyl, ethyl, carboxyl or amido are modified.But also esterification of organic molecule, alkylation and/or phosphorylation.

Term used herein " mutant " refers to compare with wild-type organisms the organism of existence sudden change in the nucleotide sequence.

Term used herein " antibody " refers to the epi-position binding fragment of Fv (sdFv), antiidiotype (anti-Id) antibody (comprising for example anti-Id antibody of anti-antibody of the present invention) and above-mentioned any antibody that monoclonal antibody, bi-specific antibody, multi-specificity antibody, people's antibody, humanized antibody, chimeric antibody, camel sourceization (camelised) antibody, single domain antibody, strand Fv (scFv), single-chain antibody, FAB fragment, F (ab ') fragment, disulfide linkage connect.Specifically, antibody comprises the immunocompetence fragment (molecule that promptly comprises antigen binding site) of immunoglobulin molecules and immunoglobulin molecules.Immunoglobulin molecules can be any type (for example IgG, IgE, IgM, IgD, IgA and IgY), class (for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Term used herein " antibody fragment " refers to the antibody fragment of immunologic opsonin in conjunction with hSARS virus or any epi-position of hSARS virus.Antibody fragment can produce by any technology well known by persons skilled in the art.For example, Fab and F (ab ') ₂Fragment can (produce F (ab ') by using enzyme such as papoid (producing the Fab fragment) or stomach en- ₂Fragment) the enzymolysis immunoglobulin molecules produces.F (ab ') ₂Fragment contains variable region, CH1 district and the hinge area of complete light chain and heavy chain.Antibody fragment also can produce by recombinant DNA technology.Antibody fragment can be one or more complementary determining regions (CDR) of antibody.

Term used herein " immunologic opsonin is in conjunction with the antibody or the antibody fragment of polypeptide of the present invention " refers to antibody or its fragment, its immunologic opsonin is in conjunction with SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its complement, analogue, derivative, fragment or its part encoded polypeptides, or immunologic opsonin is in conjunction with having SEQ ID NO:2,12,14,17-239,241-736,738-1107,1109-1589,1591-1964 or 1966-2470 aminoacid sequence or its variant, analogue, derivative or segmental polypeptide, and other polypeptide of non-specific binding not.Immunologic opsonin in conjunction with the antibody of polypeptide of the present invention or its fragment can with other antigenic cross-reaction.Preferred immunologic opsonin in conjunction with the antibody of polypeptide of the present invention or its fragment not with other antigenic cross-reaction.Immunologic opsonin can be differentiated by for example immunoassay or other technology well known by persons skilled in the art in conjunction with antibody or its fragment of polypeptide of the present invention.

Term used herein " epi-position " refers in animal, preferred mammal, most preferably has antigenicity or immunogenic hSARS virus, polypeptide or proteic fragment among the mankind.Having immunogenic epi-position is the fragment that causes the polypeptide of antibody response in animal.Having antigenic epi-position is antibody and its immunologic opsonin bonded polypeptide or proteic fragment, and this can measure by any method known in the art, for example by immunoassay as herein described.Antigenic epitopes is not that immunogenicity must be arranged.

Term used herein " antigenicity " refers to that material (for example foreign matter, microorganism, medicine, antigen, protein, peptide, polypeptide, nucleic acid, DNA, RNA etc.) causes the ability of immunne response in concrete organism, tissue and/or cell.Sometimes term " antigenic " is a synonym with term " immunogenic ".

Term used herein " immunogenicity " refers to that material (for example foreign matter, microorganism, medicine, antigen, protein, peptide, polypeptide, nucleic acid, DNA, RNA etc.) causes the characteristic of immunne response in organism.Immunogenicity depends in part on the size of the material of discussion, depends in part on this material and the dissimilar degree of host's molecule.The albumen tendency of high conservative has than reduced immunogenicity.

" isolating " nucleic acid molecule be with its natural origin in other nucleic acid molecule isolated nucleic acid molecule of existing.In addition, " isolating " nucleic acid molecule, for example the cDNA molecule can not contain other cell material or substratum substantially when producing by recombinant technology, can not contain precursor or other chemical substance substantially when synthesizing by chemical method.In a preferred embodiment of the invention, the nucleic acid molecule of code book invention polypeptides is isolating or purifying.Term " isolating " nucleic acid molecule do not comprise the member that belongs to the library, also not with the isolating nucleic acid of other library clone that contains other nucleic acid molecule.

Term used herein " hybridize under stringent condition " is described has hybridization and the wash conditions that at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% conforming nucleotide sequence keeps hybridization mutually usually each other.This hybridization conditions is described in the Biology such as but not limited to Current Protocols inMolecular, John Wiley﹠amp; Sons, N.Y. (1989), 6.3.1-6.3.6.; BasicMethods in Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y. (1986), 75-78 page or leaf and 84-87 page or leaf; And Molecular Cloning, Cold SpringHarbor Laboratory, N.Y. (1982), the 387-389 page or leaf is known for a person skilled in the art.The preferred limiting examples of stringent hybridization condition is to hybridize in about 68 ℃ of following 6X sodium chloride/sodium citrate (SSC), 0.5%SDS, at room temperature washs one or many among 2X SSC, the 0.5%SDS then.The preferred limiting examples of another of stringent hybridization condition is to hybridize in about 45 ℃ of following 6X SSC, washs one or many then in about 50-65 ℃ of following 0.2XSSC, 0.1%SDS.

" isolating " or " purifying " peptide or albumen do not contain substantially from described proteinic cell source or tissue-derived cell material or other contaminating protein matter, or when they are synthetic by chemical method, do not contain precursor or other chemical substance substantially.Word " does not contain cell material substantially " and comprises the polypeptides goods, and wherein polypeptides is separated from the cellular constituent of the cell of its separation or reorganization production.Therefore, the polypeptides that does not contain cell material substantially comprises and contains the polypeptides goods that are less than about 30%, 20%, 10%, 5%, 2.5% or 1% (dry weight) contaminating protein matter.When polypeptides is recombinant production, also preferably do not contain substratum substantially, it is about 20% that the volume of promptly cultivating the fiduciary point protein articles is less than,, 10% or 5%.When polypeptides is produced by chemosynthesis, preferably do not contain precursor or other chemical substance substantially, promptly separate with the precursor or other chemical substance that are used to carry out protein synthesis.Therefore, this polypeptides goods contain and are less than precursor or the compound that about 30%, 20%, 10%, 5% (dry weight) do not belong to desired polypeptides/protein fragments.In the preferred embodiment of the invention, polypeptides is isolating or purifying.

Term used herein " isolating " virus be with its natural origin in the isolating virus of other organism that exists, described source for example is biomaterial such as cell, blood, serum, blood plasma, saliva, urine, excrement, phlegm, nasopharynx aspirate etc.Isolating virus can be used for infecting object.

Term used herein " has polypeptide biological activity of the present invention " and refers to have common bioactive polypeptide or proteinic characteristic, and it is compared with following polypeptide has similar or same structure territory and/or have enough amino acid consistence: SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its complement, analogue, derivative, fragment or part encoded polypeptides; Or have SEQ ID NO:2,12,14,17-239,241-736,738-1107,1109-1589,1591-1964 or 1966-2470 aminoacid sequence or its variant, analogue, derivative or a segmental polypeptide.This common biological activity of polypeptide of the present invention comprises antigenicity and immunogenicity.

Term used herein " part " or " fragment " refer to contain the associated nucleic acid molecular length at least about 25,30,35,40,45,50,60,70,80,90,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1,000,1,050,1,100,1,150,1,200,2,000,3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,11,000,12,000,13,000,14,000,15,000,16,000,17,000,18,000,19,000,20,000,21,000,22,000,23,000,24,000,25,000,26,000,27,000,28,000,29,000 or more a plurality of continuous nucleic acid, and have the nucleic acid molecule fragment of at least a functional character (or its encoded protein matter has the coded proteinic a kind of functional character of associated nucleic acid molecule) of associated nucleic acid molecule; Or refer to contain at least 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,90,100,120,140,160,180,200,220,240,260,280,300,320,340,360,400,500,600,800,1 of related protein or polypeptide length, 000,2,000,3,000,4,000,5,000,6,000,7,000,8,000,9,000,9,500 or more a plurality of amino-acid residue, and have the protein or the polypeptide fragment of at least a functional character of related protein or polypeptide.

It has and second kind of similar or identical functions of protein matter term used herein " analogue " for protein matter (for example albumen, polypeptide, peptide and antibody) finger protein class material, but not to comprise and second kind of aminoacid sequence that protein matter is similar or identical, or have and second kind of structure that protein matter is similar or identical.In a specific embodiments, the source antibody mediated immunity specificity of antibody analog and this analogue is in conjunction with identical epi-position.In another embodiment, antibody analog and the source antibody mediated immunity specificity of this analogue are in conjunction with different epi-positions.Protein matter with similar aminoacid sequence refers to satisfy second kind of protein matter of at least one following condition: (a) its aminoacid sequence of protein matter is identical with the aminoacid sequence at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of second kind of protein matter; (b) its coding nucleotide sequence of protein matter under stringent condition with nucleotide sequence hybridization, at least 5 continuous amino acid residues of described nucleotide sequence coded second kind of protein matter, at least 10 continuous amino acid residues, at least 15 continuous amino acid residues, at least 20 continuous amino acid residues, at least 25 continuous amino acid residues, at least 40 continuous amino acid residues, at least 50 continuous amino acid residues, at least 60 continuous amino acid residues, at least 70 continuous amino acid residues, at least 80 continuous amino acid residues, at least 90 continuous amino acid residues, at least 100 continuous amino acid residues, at least 125 continuous amino acid residues or at least 150 continuous amino acid residues; (c) nucleotide sequence at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of second kind of protein matter of its coding nucleotide sequence of protein matter and coding is identical.The protein matter that has analog structure with second kind of protein matter refers to have with second kind of protein matter the protein matter of similar secondary, three grades or quaternary structure.The structure of protein matter can be measured by method known to those skilled in the art, includes but not limited to peptide sequencing, X-ray crystallography, nucleus magnetic resonance, circular dichroism and crystal current sub-microscope.

For measuring the consistence per-cent of two seed amino acid sequences or two kinds of nucleotide sequences, for reaching best relatively purpose aligned sequence (for example in the first seed amino acid sequence or nucleotide sequence, introducing the room) to reach best alignment with second seed amino acid or nucleotide sequence.Then at corresponding amino acid position or nucleotide position comparing amino acid residue or Nucleotide.When the position of first kind of sequence was occupied by the amino acid identical with second kind of sequence corresponding position or Nucleotide, described molecule was identical in this position.Consistence per-cent between two kinds of sequences is the function (being quantity/total number of positions amount * 100% of % consistence=identical lap position) of the quantity of the total same position of two kinds of sequences.In one embodiment, two kinds of sequences have equal length.

The mensuration of consistence per-cent can use mathematical algorithm to finish between two kinds of sequences.The preferred limiting examples that is used for the mathematical algorithm of two kinds of sequences of comparison is Karlin and Altschul, 1990, the algorithm of Proc.Natl.Acad.Sci.U.S.A.87:22642268, at Karlin and Altschul, 1993, be modified among the Proc.Natl.Acad.Sci.U.S.A.90:58735877.This algorithm is incorporated into Altschul etc., and 1990, among the NBLAST and blast program of J.Mol.Biol.215:403.BLAST Nucleotide is retrieved available NBLAST Nucleotide program and is carried out, and parameter setting is in for example score value=100, and word length=12 are to obtain and nucleic acid molecule homologous nucleotide sequence of the present invention.BLAST albumen is retrieved available XBLAST program and is carried out, and parameter setting is in for example score value=50, and word length=3 are to obtain and protein molecule homologous aminoacid sequence of the present invention.For obtaining to be used for the alignment of having vacant position of comparison, can use Altschul etc., 1997, the Gapped BLAST described in the Nucleic Acids Res.25:33893402.Perhaps, can use PSI BLAST to carry out between detection molecules the search that iterates of edge relation far away (Id.).When using BLAST, Gapped BLAST and PSI Blast program, can use the parameter (referring to for example NCBI website) of each program (for example XBLAST and NBLAST) acquiescence.Another the preferred limiting examples that is used for the mathematical algorithm of comparative sequences is Myers and Miller, 1988, and the algorithm of CABIOS 4:1117.This algorithm is incorporated in the ALIGN program (2.0 editions), and it is the part of GCG sequence alignment software package.When using ALIGN program comparing amino acid sequence, can use PAM120 weighting residue table, room length point penalty 12 and gap penalty 4.

Consistence per-cent can use the technical measurement similar to above-mentioned technology between two kinds of sequences, allows or do not allow the room.When calculating consistence per-cent, the general coupling fully of only calculating.

Term used herein " derivative " refers to comprise the protein matter of the aminoacid sequence that changes by introducing radical amino acid replacement, disappearance and/or insertion for protein matter (for example protein, polypeptide, peptide and antibody).Term used herein " derivative " also refers to adorned protein matter, promptly by the molecule of any kind is covalently bound to protein matter.For example but never in any form restriction, antibody can be modified, for example by glycosylation, acetylize, PEGization, phosphorylation, amidation, derivation, proteolysis, connection cell ligand or other albumen etc. by known protection/blocking groups.The derivative of protein matter can produce by using the chemically modified of those skilled in the art's known technology, and described technology includes but not limited to that the metabolism of specificity chemical cracking, acetylize, formylation, tunicamycin is synthesized.In addition, the derivative of protein matter can comprise one or more atypia amino acid.The derivative of protein matter has or identical functions similar to the protein matter in its source.

Term used herein " object " and " patient " are used interchangeably.Term used herein " object " refers to animal, preferably include non-primate (for example ox, pig, horse, goat, sheep, cat, dog, birds and rodents) and primate (for example monkey such as macaque (cynomolgous monkey) and people's) Mammals, more preferably the people.

4. accompanying drawing summary

Fig. 1 shows that the RNA RNA-dependent polymerase protein of described SARS virus and known coronavirus has 57% homology from the partial dna sequence (SEQ ID NO:1) of SARS virus acquisition and the aminoacid sequence (SEQ ID NO:2) of inferring thereof.

Fig. 2 shows the electron photomicrograph of the novel hSARS virus of the morphological feature with similar coronavirus.

Fig. 3 shows the immunofluorescence dyeing with the FrHK-4 cell bonded IgG antibody that infects novel coronavirus section (Coronaviridae) human respiratory virus.

Fig. 4 show grow in the cell culture, at the electron photomicrograph of pH7.0 with the ultracentrifugal sedimentation thing of the hSARS virus of 3% phospho-wolframic acid potassium negative staining.

Fig. 5 A shows the thin layer section electron photomicrograph of SARS patient's lung tissue biopsy; Fig. 5 B shows the thin layer section electron photomicrograph of hSARS virus infected cell.

Fig. 6 shows Partial Protein sequence (215 amino acid of hSARS virus (GenBank searching number AY268070); SEQ ID NO:2) phylogenetics analytical results.Genealogical tree makes up by adjacent method.Horizon distance is represented the number in the site that two sequences of comparing are different.The bootstrap value is inferred from 500 repetitions and is drawn.

Fig. 7 A be presented at can detection by quantitative in the sample in the real-time quantitative PCR test of hSARS virus fluorescence intensity to PCR round-robin amplification figure.Pointed out the copy number of input plasmid DNA in the reaction.X-axis is represented the cycle number of quantitative PCR test, and Y-axis represents to surpass the fluorescence intensity (FI) of background.Fig. 7 B shows the curve analysis result of the PCR product of clinical sample.Pointed out to derive from the positive (+ve) sample, the feminine gender (ve) signal of sample and water contrast (water).X-axis represent temperature (℃, Y-axis represents to surpass the fluorescence intensity (FI) of background.

Fig. 8 shows another kind of from the partial dna sequence (SEQ IDNO:11) of hSARS virus acquisition and the aminoacid sequence (SEQ ID NO:12) of inferring thereof.

Fig. 9 shows that another is from the partial dna sequence (SEQ IDNO:13) of hSARS virus acquisition and the aminoacid sequence (SEQ ID NO:14) of inferring thereof.

Figure 10 shows the complete genome group dna sequence dna (SEQ ID NO:15) of hSARS virus.

Figure 11 shows the deduction aminoacid sequence that obtains from SEQID NO:15 with three kinds of frames (referring to SEQ ID NO:16,240 and 737).The terminator codon of the end of asterisk (*) indicator sign peptide.The first frame aminoacid sequence: SEQ ID NO:17-239; The second frame aminoacid sequence: SEQ ID NO:241-736; Third reading frame aminoacid sequence: SEQ ID NO:738-1107.

Figure 12 shows with the deduction aminoacid sequence of three kinds of frames (referring to SEQ ID NO:1108,1590 and 1965) from the complement acquisition of SEQ ID NO:15.The terminator codon of the end of asterisk (*) indicator sign peptide.The first frame aminoacid sequence: SEQ ID NO:1109-1589; The second frame aminoacid sequence: SEQ ID NO:1591-1964; Third reading frame aminoacid sequence: SEQ ID NO:1966-2470.

Figure 13 shows the nucleotide sequence of forward primer (SEQ ID NO:2471 and 2474), reverse primer (SEQ IDNO:2472 and 2475) and hybridization probe (SEQ ID NO:2473 and 2476), is used for quantitatively Test is to measure hSARS virus.

Figure 14 shows the typical curve that is used for real-time quantitative RT-PCR test SAS-CoV.Threshold cycle number (Ct) is that the reaction fluorescence intensity reaches the required PCR cycle number of predetermined threshold.The logarithm of Ct and plasmid DNA initial concentration is inversely proportional to.Provided relation conefficient.In standard amplification figure, the initial copy number of difference is calculated Ct based on calculated threshold by the maximum curvature method.The X-axle is represented the logarithm of standard substance copy number, and the Y-axle is represented Ct.

Figure 15 shows and to use modification RT-PCR detection method of the present invention for from the isolating NPA sample of SARS patient, with fluorescence intensity to the typical case of the PCR cycle number work figure that increases.By the RNA extracting method of revising, 40 in the 50NPA sample is male in test in real time.For in first-generation RT-PCR test, being negative sample, find wherein all to contain very a spot of viral RNA by detection method of the present invention.The X-axle is represented the PCR cycle number, and the Y-axle represents to surpass the fluorescence intensity (Δ Rn) of background.

Figure 16 shows the viral load amount of SARS-CoV in the clinical sample and the graph of a relation of Start Date.The result shows that the viral load amount increases with progression of disease.Some positive sample in first-generation test is found and contains very a large amount of viral RNAs.The X-axle is represented Start Date, and the Y-axle is represented the copy number of each reaction in the sample.

5. detailed Description Of The Invention

The purposes of sequence information in diagnostic method of the hSARS virus that the present invention relates to separate. Specifically, the invention provides the method that whether detection hSARS nucleic acid molecules viral or its natural or artificial variant, analog or derivative exists in biological sample. Described method comprises from various sources acquisition biological samples, sample contact with compound or the reagent of the nucleic acid (for example mRNA, geneome RNA) that can detect hSARS virus or its natural or artificial variant, analog, derivative, thus the existence of viral or its natural or artificial variant of hSARS, analog, derivative in the test sample. The reagent of preferred detection hSARS mRNA or geneome RNA be can with the labeling nucleic acid probe of mRNA or geneome RNA hybridization. In a preferred embodiment, described nucleic acid probe is the nucleic acid molecules that comprises or be made up of SEQ ID NO:2473 or 2476 nucleotide sequences or its part, and it is enough to specificity and hSARS mRNA or geneome RNA hybridization under stringent condition. In a preferred specific embodiments, in sample, pass through RT-polymerase chain reaction (RT-PCR), the primer that use makes up based on part hSARS viral nucleotide sequences detects the existence of hSARS virus or its natural or artificial variant, analog or derivative. In a non-limiting specific embodiments, the primer that uses in the preferred RT-PCR method is:

5 '-CAGAACGCTGTAGCTTCAAAAATCT-3 ' (SEQ ID NO:2471) and

5′-TCAGAACCCTGTGATGAATCAACAG-3′(SEQ ID NO：2472)，

At MgCl₂Exist lower, thermal cycle for example for but be not limited to 50 ℃ 2 minutes, 95 ℃ 10 minutes, then 95 ℃ of 45 of 15 seconds, 60 ℃ circulations of 1 minute (also referring to following 6.7,6.8,6.9 joints). In preferred embodiments, described primer comprises SEQ ID NO:2471 and 2472 nucleotide sequences. In another non-limiting specific embodiments, the primer that uses in the preferred RT-PCR method is: 5 '-ACCAGAATGGAGGACGCAATG-3 ' (SEQ IDNO:2474) and 5 '-GCTGTGAACCAAGACGCAGTATTAT-3 ' (SEQ IDNO:2475), at MgCl₂Exist lower, thermal cycle for example for but be not limited to 50 ℃ 2 minutes, 95 ℃ 10 minutes, then 95 ℃ of 45 of 15 seconds, 60 ℃ circulations of 1 minute (also referring to following 6.7,6.8,6.9 joints). In preferred embodiments, described primer comprises SEQ ID NO:2474 and 2475 nucleotide sequences.

The inventive method can comprise the real-time quantitative PCR test. In a preferred embodiment, the quantitative PCR that uses among the present invention is

Test (Holland etc., Proc Natl AcadSci USA 88 (16): 7276 (1991)). This mensuration can carried out for the instrument that carries out this test, for example can be available from the instrument of Applied Biosystems (Foster City, CA). In preferred specific embodiments, the invention provides the real-time quantitative PCR test, by making from the total RNA reverse transcription of the extraction of sample, the cDNA that obtains is carried out the PCR reaction with Auele Specific Primer, use the existence of probe in detecting amplified production, thereby in biological sample, detect the existence of hSARS virus or its natural or artificial variant, analog or derivative. In preferred embodiments, described probe is

Dark pin, its by with 5 '-report dyestuff and 3 '-oligonucleotides of quencher dyestuff forms. In a preferred embodiment, described probe have 5 '-nucleotide sequence of TCTGCGTAGGCAATCC-3 ' (SEQ ID NO:2473). In a further preferred embodiment, described probe have 5 '-nucleotide sequence of ACCCCAAGGTTTA-CCC-3 ' (SEQ D NO:2476). Fluorescence report dyestuff, for exampleDyestuff, covalently bound with 5 of oligonucleotide probe ' end. Other dyestuff, for exampleDyestuff or VIC can be used as the report dyestuff. Each reports that dyestuff is by 3 ' end

Dyestuff or non-fluorescence quencher quencher. In a preferred embodiment, 3 ' end NFQ-MGB mark. Because the thermal cycle through some has produced the PCR product, prolonging the fluorescence signal of catching these reactions when step finishes, therefore can be based on viral load amount in the amplification figure quantitative assay sample.

Also can use other to detect the technology of RNA. For example, the ex vivo technique of detection mRNA comprises Northern hybridization, in situ hybridization, RT-PCR and RNA enzyme protection. The ex vivo technique that detects geneome RNA comprises Northern hybridization, RT-PCT and RNA enzyme protection.

As mentioned above, in a preferred embodiment, the polynucleotides of hSARS virus can amplification before it is detected. Term " amplification " refers to prepare from single polynucleotide molecule the process of a plurality of copies of this nucleic acid. The amplification of polynucleotides can be undertaken by biochemical method well known by persons skilled in the art external. Amplifing reagent can be any compound or system that primer prolongs the product complex functionality of finishing, and comprises enzyme. The suitable enzymes that is used for this purpose comprises Klenow fragment, T4DNA polymerase, other obtainable archaeal dna polymerase, polymerase mutation albumen, reverse transcriptase, the ligase of for example e. coli dna polymerase I, Taq polymerase, e. coli dna polymerase I and comprises other enzyme of heat-stabilized enzyme (namely carrying out the enzyme that primer prolongs after being elevated to the temperature that is enough to cause sex change). Suitable enzymatic advances nucleotides with suitable mode combination, with the primer prolongation product of formation with each mutant nucleotide chain complementation. In a preferred embodiment, described enzyme is the AmpliTaq available from Applied BiosystemsArchaeal dna polymerase. Usually, this synthetic from 3 of each primer '-end, along template strand to 5 '-direction is carried out, until end of synthesis, the molecule of generation different length. But some amplifing reagents uses process same as described above, 5 '-end begins to synthesize, carries out to other direction. Under any circumstance, method of the present invention is not limited to the specific embodiments of amplification described herein.

The spendable a kind of amplification in vitro method of the present invention is United States Patent (USP) 4,683,202 and 4,683, and the polymerase chain reaction described in 195 (PCR). Term " polymerase chain reaction " refers to use the method for heat-stable DNA polymerase and two kinds of Oligonucleolide primers DNA amplification base sequences, described primer a kind of an end and the complementation of (+)-chain in sequence to be amplified, another kind of in the other end and the complementation of (-)-chain. Because new synthetic DNA chain can be subsequently as the extra template of same primers as sequence, primer annealing, chain elongation and the continuous circulation that dissociates make required sequence generation fast and the high specific amplification. Polymerase chain reaction is used for detecting at sample the existence of the polynucleotides of the Codocyte factor. Many polymerase chain methods are well known by persons skilled in the art, can be used for method of the present invention. For example, DNA can experience 30-35 following amplification cycles in thermal cycler: 95 ℃ of 30 seconds, 52-60 ℃ 1 minute and 72 ℃ 1 minute, finally prolong step and be 72 ℃ 5 minutes. Again for example, DNA can experience the circulation of 35 polymerase chain reactions in thermal cycler, denaturation temperature be 95 ℃ 30 seconds, then variable annealing temperature 54-58 ℃ 1 minute, prolong step and be 70 ℃ 1 minute, finally prolong step and be 70 ℃ 5 minutes.

The primer that is used for amplification hSARS virus mRNA or geneome RNA can use any suitable method preparation, and for example traditional phosphotriester and di-phosphate ester method or its automation embodiment are as long as primer can be hybridized with herbicide-tolerant polynucleotide. Method such as the United States Patent (USP) 4,458 of synthetic oligonucleotide on a kind of solid carrier modifying are described in 066. The exact length of primer depends on many factors, comprises that temperature, buffer solution and nucleotides form. Primer must be induced to start in the presence of the reagent in amplification and be prolonged the synthetic of product.

The primer that the inventive method is used and each chain complementation of nucleotide sequence to be amplified. Term " complementation " guides thing allowing reagent to carry out under the condition of polymerization and their chain hybridization separately. In other words, primer and flanking sequence hybridization with the flanking sequence complementation allow the amplification of nucleotide acid sequence. The primer 3 of preferred continuity ' end has the complete base pairing complementarity with side chain complementation. Useful known method is developed primer and probe for hSARS virus polynucleotides in conjunction with the disclosure. In preferred embodiments, primer basis

Primer scheme (Applied Biosystems) design. Described primer uses the Primer Express Software for Design described in the Primer Express UserBulletin (Applied Biosystems). Briefly, when the design primer, it should be selected behind probe. Primer is preferably as far as possible approaching and not overlapping with probe with probe. The G-C content of primer should be in the scope of 20%-80%. Preferably avoid a plurality of identical nucleotides. Especially for guanine, preferably avoid 4 or how continuous G. The melting temperature of each primer is preferably 58 ℃-60 ℃. Preferred G and/or C base are no more than 2 in 5 nucleotides of each primer 3 ' end.

Can use the Primer Express Software for Design probe of Primer Express User Bulletin (P/N 4317594) described in (AppliedBiosystems). Briefly, preferably keep G-C content in the scope of 20%-80%. Preferably avoid a plurality of identical nucleotides. Especially for guanine, preferably avoid 4 or how continuous G. Preferably the G base is not placed on 5 ' end. The preferred chain of selecting to make C in the probe to surpass G. Preferred two kinds of probes are on same chain. For single probe test, melting temperature is preferably 68 ℃-70 ℃.

Those of ordinary skills understand the various amplification methods that can be used for increasing the target nucleic acid copy number. The polynucleotides that detect in the inventive method further are evaluated after can or being attached to solid carrier in solution; detect; the clone; order-checking etc.; this can be by any method that is generally used for detecting concrete nucleotide sequence; such as another polymerase chain reaction; oligomer restriction enzyme digestion (Saiki etc.; Bio/Technology 3:1008-1012 (1985)); allele specific oligonucleotide (ASO) probe analysis (Conner etc.; Proc.Natl.Acad Sci.USA 80:278 (1983); oligonucleotides joint test (OLA) (Landegren etc., Science 241:1077 (1988)); RNA enzyme protection test etc. The summary (Landegren etc., Science 242:229-237 (1988)) of the molecular engineering of existing DNA analysis. After the DNA cloning, product detects by the DNA hybridization analysis, and does not use radioactive probe. In this method, for example, a small amount of DNA sample that contains the polynucleotides that obtain from tissue or object is amplified, by the southern blotting technique technical Analysis. The height amplifying signal has promoted the use of nonradioactive probe or mark. In one embodiment of the invention, a kind of NTP is by radioactive label, thereby permission directly shows amplified production by autoradiography. In another embodiment, amplimer is by fluorescence labeling, and electrophoresis is by the electrophoresis system. Show rather than radiated signal by area of computer aided figure after the laser detection, show amplified production.

The primer that is used for the part of amplification hSARS virus mRNA or geneome RNA, its size is at least 10,15,20,25 or 30 length of nucleotides. Preferred GC ratio should be higher than 30%, 35%, 40%, 45%, 50%, 55% or 60%, to prevent the hairpin structure on the primer. In addition, amplicon should have enough length to detect by the standard molecular biology method. Preferred amplicon length is at least 20,30,40,50,60,70,80,90,100,110,120,130,140,150,175,200,250,300,350,400,450,500,550,600,700,800 or 1000 base-pairs.

In a specific embodiments, the inventive method further relates to from contrast object acquisition control sample, with control sample with can test sample in mRNA or the geneome RNA compound or the reagent that exist contact, and mRNA in the control sample or geneome RNA existed with mRNA or genomic DNA existence in the testing sample compare.

The present invention also is included in and detects the kit that the hSARS viral nucleic acid exists in the testing sample. This kit for example can comprise labeled compound or the reagent that can detect nucleic acid molecules in testing sample, in certain embodiments the instrument (in conjunction with the oligonucleotide probe of DNA or mRNA) of mRNA amount in the inclusion test sample.

For the kit based on oligonucleotides, this kit can for example comprise: (1) oligonucleotides, detectable labeled oligonucleotide for example, the nucleotide sequence hybridization of itself and hSARS virus; And/or (2) are used for the primer pair that amplification contains the nucleic acid molecules of hSARS virus sequence. This kit also can comprise, for example, and buffer reagent, anticorrisive agent or protein stabilized reagent. But this kit also inclusion test can detect the necessary composition of thing (for example enzyme or substrate). This kit also can comprise control sample or a series of control sample, and it can be detected and compare with testing sample. The various compositions of this kit are encapsulated into independent container usually, and all various containers and operation instruction are enclosed in the individual packaging thing together.

5.1hSARS the nucleotide sequence of virus

The purposes of the sequence information of the virus that the present invention relates to separate in diagnosis and methods for the treatment of. The complete genome group sequence of hSARS virus CCTCC-V200303 is disclosed in the U.S. Patent application of submitting to simultaneously with this paper on March 24th, 2004, lawyer's file number V9661.0069, its by reference integral body be attached to herein. In a specific embodiments, the invention provides complete nucleotide sequence SEQ ID NO:15 or its complement, analog, derivative, fragment or the part of hSARS virus CCTCC-V200303. In addition, the present invention relates under stringent condition nucleic acid molecules with any part hybridization of hSARS virus CCTCC-V200303 genome SEQ ID NO:15. In a specific embodiments, the invention provides the nucleic acid molecules that is suitable as primer, this nucleic acid molecules comprises or is made up of SEQ ID NO:1,3,4,11 or 13 nucleotide sequences or its complement, analog, derivative, fragment or part. In preferred specific embodiments, described primer comprises SEQ ID NO:2471,2472,2474 or 2475 nucleotide sequences. In another embodiment, the invention provides the nucleic acid molecules that is suitable as hybridization probe, for detection of comprising or by SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its complement, analog, derivative, fragment or part nucleic acid that form, code book invention polypeptide. In another embodiment, the present invention relates to comprise the kit that its nucleotide sequence is the primer of SEQ ID NO:2471 and/or 2472, to detect hSARS virus or its natural or artificial variant, analog or derivative. In a preferred embodiment, described kit also comprises the probe with SEQ ID NO:2473 nucleotide sequence. In another embodiment, the present invention relates to comprise the kit that its nucleotide sequence is the primer of SEQ ID NO:2474 and/or 2475, to detect hSARS virus or its natural or artificial variant, analog or derivative. In a preferred embodiment, described kit also comprises the probe with SEQ ID NO:2476 nucleotide sequence. In a further preferred embodiment, described kit also comprises reagent, for detection of as non-existent gene in the hSARS virus of negative control. The present invention further comprises embedded virus or recombinant virus or by described nucleotide sequence coded virus protein.

The present invention also relates to the nucleic acid molecules of the separation of hSARS virus, described nucleic acid molecules comprises or is made up of SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its complement, analog, derivative, fragment or part. In another embodiment, the invention provides the isolated nucleic acid molecule of under the stringent condition of this paper definition, hybridizing with the nucleic acid molecules with SEQ ID NO:1,11,15,13,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or the known member's of coronaviridae specific gene or its complement, analog, derivative, fragment or part. In another embodiment, the invention provides isolated polypeptide or the protein of nucleic acid molecule encoding, described nucleic acid molecules comprise SEQ ID NO:1 nucleotide sequence or its complement, analog, derivative or fragment at least about 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600 or the nucleotide sequence of more continuous nucleotides. In another embodiment, the invention provides isolated polypeptide or the protein of nucleic acid molecule encoding, described nucleic acid molecules comprise SEQ IDNO:11 nucleotide sequence or its complement, analog, derivative or fragment at least about 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200 or the nucleotide sequence of more continuous nucleotides. In another specific embodiments, the invention provides isolated polypeptide or the protein of nucleic acid molecule encoding, described nucleic acid molecules comprise SEQ ID NO:13 nucleotide sequence or its complement, analog, derivative or fragment at least about 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700 or the nucleotide sequence of more continuous nucleotides. In another specific embodiments, the invention provides isolated polypeptide or the protein of nucleic acid molecule encoding, described nucleic acid molecules comprises SEQ ID NO:15 nucleotide sequence or its complement, analog, derivative or fragment at least about 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200,2000,3000,4000,5000,6000,7000,8000,9000,10000,11000,12000,13000,14000,15000,16000,17000,18000,19000,20000,21000,22000,23000,24000,25000,26000,27000,28000,29000 or the nucleotide sequence of more continuous nucleotides. Polypeptide comprises the polypeptide shown in Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and 12 (SEQ ID NO:1109-1589,1591-1964 and the 1966-2470). Polypeptide of the present invention or albumen preferably have one or more biologically actives of following protein: by the protein of SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleic acid sequence encodings; Or contain natural viral protein by the amino acid sequence of SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleic acid sequence encodings.

The present invention further provides the energy specific binding by the polypeptide of the present invention of SEQ ID NO:1,11,13,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its fragment coding or the antibody of any hSARS epi-position. The present invention also provides the polypeptide of the present invention of specific binding SEQ ID NO:15 nucleotide sequence or its fragment coding or the antibody of any hSARS epi-position. This antibody includes but not limited to polyclonal antibody, monoclonal antibody, bispecific antibody, multi-specificity antibody, people's antibody, humanized antibody, chimeric antibody, single-chain antibody, Fab fragment, F (ab ')₂The VL that Fy, the intracellular antibody that fragment, disulfide bond connect and containing can be combined with polypeptid specificity of the present invention or the fragment of VH domain and even complementary determining region (CDR).

In another embodiment, the invention provides the vaccine product that comprises hSARS virus or its natural or artificial variant, analog or derivative. In another embodiment, the invention provides the recombinant forms that comprises hSARS virus and the vaccine product of chimeric form or viral sub-units. In a specific embodiments, vaccine product of the present invention comprises alive but the hSARS virus of attenuation is with or without medicine and can accepts excipient, comprises adjuvant. In another embodiment, vaccine product of the present invention comprises deactivation or dead hSARS virus, is with or without drug acceptable carrier, comprises adjuvant. Vaccine product of the present invention also can comprise adjuvant. Therefore, the present invention further provides the recombinant forms of preparation hSARS virus or the method for chimeric form. In another embodiment, vaccine product of the present invention comprises one or more nucleic acid molecules that comprises or be made up of SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its fragment. In another embodiment, the invention provides the vaccine product that comprises one or more polypeptide of the present invention, described polypeptide by comprise or by SEQ ID NO:1,11,13,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its fragment form nucleotide sequence coded. In another embodiment, the invention provides the vaccine product that comprises one or more polypeptide of the present invention, described polypeptide by comprise or by SEQ ID NO:15 nucleotide sequence or its fragment form nucleotide sequence coded. In addition, the invention provides by medication combinedly giving vaccine product of the present invention or antibody is treated separately or with following, improve, the method of control or prevention SARS: antivirotic [amantadine for example, Rimantadine, GCV, ACV, Ribavirin, Penciclovir, oseltamivir, FOSCARNET, Zidovudine (AZT), Didanosine (ddI), Lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), NVP, delavirdine, indinavir, Ritonavir, arabinosy ladenosine, viracept see nelfinaivr, inverase, zanamivir, oseltamivir phosphate, pleconaril, interferon etc.], steroids and corticosteroid are (such as metacortandracin, cortisone, fluticasone) and glucocorticoid, antibiotic, anodyne, bronchodilators or other are used for the medicine of respiratory tract and/or virus infections.

In addition, the invention provides the pharmaceutical composition that comprises antivirotic of the present invention and drug acceptable carrier. The present invention also provides the medicine box that comprises pharmaceutical composition of the present invention.

On the other hand, the invention provides that screening can suppress hSARS virus or its natural or artificial variant, analog or derivative infectiousness or the method for the antivirotic that copies.

In one embodiment, the invention provides and in biomaterial such as cell, blood, serum, blood plasma, saliva, urine, excrement, phlegm, nasopharynx aspirate etc., detect hSARS virus of the present invention or its natural or artificial variant, analog or derivative exist, the active or method expressed. Can directly or indirectly detect by biomaterial is contacted the reagent of the existence of hSARS virus or its natural or artificial variant, analog or derivative, the existence of hSARS virus or its natural or artificial variant, analog or derivative in the working sample. In a specific embodiments, detecting reagent is antibody of the present invention. In another embodiment, detecting reagent is nucleic acid of the present invention.

5.2.hSARS virus

5.2.1.hSARS the natural variant of virus

The present invention is based on the inventor to separation and evaluation from the new virus of suffering from the SARS object. The hSARS virus of separating is deposited in Chinese Typical Representative culture center (CCTCC) on April 2nd, 2003, is given searching number CCTCC-V200303. The present invention also relates to the natural variant of the hSARS virus of preservation searching number CCTCC-V200303.

The natural variant of hSARS virus has the sequence different from the hSARS virus genome sequence, this is because one or more spontaneous mutations of genome sequence, include but not limited to point mutation, rearrangement, insertion, disappearance etc., described sudden change meeting or can not cause taking place phenotypic alternation. Preferred described variant comprises with respect to hSARS virus and is less than 25,20,15,10,5,4,3 or 2 replacement of amino acids, rearrangement, insertion and/or disappearances.

Conservative or non-conservative amino acid replacement can carry out at one or more amino acid residues. In preferred embodiments, variant has conservative amino acid replacement at the nonessential amino acid residue (namely expression active for viral organism as infectivity, replication capacity, albumen synthesis capability, assemble ability and CPE is not conclusive amino acid residue) of one or more predictions. In other embodiments, variant has non-conservative amino acid replacement at the nonessential amino acid residue (namely expression active for viral organism as infectivity, replication capacity, albumen synthesis capability, assemble ability and CPE is not conclusive amino acid residue) of one or more predictions.

" conservative amino acid replacement " be wherein amino acid residue by the similar radical amino acid replacement of side-chain charges. " non-conservative amino acid replacement " be wherein amino acid residue by the opposite radical amino acid replacement of side-chain charges. The family of the amino acid residue that side-chain charges is similar is defined in this area. The amino acid of genetic coding can be divided into four families: (1) acidity=aspartic acid, glutamic acid; (2) alkalescence=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) no charge polarity=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In a similar manner, all amino acid can be classified as (1) acidity=aspartic acid, glutamic acid; (2) alkalescence=lysine, arginine, histidine; (3) aliphatic series=glycine, alanine, valine, leucine, isoleucine, serine, threonine, serine and threonine are optional to be categorized as separately aliphatic series-hydroxyl; (4) aromatics=phenylalanine, tyrosine, tryptophan; (5) acid amides=asparagine, glutamine; (6) sulfur-bearing=cysteine and methionine. (referring to for example Biochemistry, the 4th edition, L.Stryer, WH Freeman and Co compile: 1995).

The invention further relates to the hSARS virus mutant. In one embodiment, sudden change can be introduced at random along all or part coded sequence of hSARS virus or its variant, for example by saturation mutagenesis, can screen the biologically active of gained mutant to identify the mutant of retentive activity. Also can use induced-mutation technique known in the art, include but not limited to rite-directed mutagenesis, mutagenesis, Site direct mutagenesis, use such as QuikChange direct mutagenesis kit (Stratagene) etc. The limiting examples of this modification comprises that amino acid replacement is that cysteine forms disulfide bond; Amino acid replacement be tyrosine and subsequently the chemical treatment polypeptide to form the dityrosine key, as in detail disclosed herein; One or more amino acid replacements and/or biological or chemical modify to produce little molecule (substrate or inhibitor) binding pocket; And/or introducing side chain specific marker (for example to differentiate interaction of molecules or capture protein-protein interaction companion (protein-protein interaction partner)). In a specific embodiments, bio-modification comprises alkylation, phosphorylation, sulphation, oxidation or reduction, ADP-ribosylation, hydroxylating, glycosylation, the addition of glucose phosphatidylinositols, ubiquitin. In another embodiment, chemical modification comprises the electric charge that changes recombinant virus. In another embodiment, positive charge or negative electrical charge are joined in the amino acid residue by chemistry, and wherein charged amino acid residue is modified to uncharged residue.

5.2.2. restructuring and chimeric hSARS virus

The present invention comprises that also origin comes from the reorganization or the embedded virus of the genomic virus vector coding of hSARS virus or its natural variant.In a specific embodiments, recombinant virus derives from the hSARS virus that preserving number is CCTCC-V200303.In a specific embodiments, virus has SEQ ID NO:15 nucleotide sequence.In another embodiment, recombinant virus derives from the natural variant of hSARS virus.The natural variant of hSARS virus has the different sequence of genome sequence (SEQ IDNO:15) with hSARS virus CCTCC-V200303, this is because one or more spontaneous mutations of genome sequence, include but not limited to point mutation, rearrangement, insertion, disappearance, displacement etc., described sudden change meeting or can not cause taking place phenotypic alternation.According to the present invention, derive from the genomic virus vector of hSARS virus CCTCC-V200303 and contain the nucleotide sequence of the part of an ORF of hSARS virus at least of encoding.In a specific embodiments, described OFR comprises or is made up of SEQ IDNO:1,11 or 13 nucleotide sequences or its fragment.In a specific embodiments, in SEQ ID NO:15 nucleotide sequence or its fragment, there is OFR, as shown in Figure 11 (SEQ ID NO:16,240 and 737) and Figure 12 (SEQ ID NO:1108,1590 and 1965) more than one.In another embodiment, the ORF encoded polypeptides comprise or by SEQ IDNO:2,12 or 14 aminoacid sequences or its fragment or as shown in Figure 11 (referring to SEQ ID NO:17-239,241-736 or 738-1107) and Figure 12 (referring to SEQ ID NO:1109-1589,1591-1964 or 1966-2470) polypeptide or its fragment form.According to the present invention, these virus vector can comprise or not comprise and are not the genomic nucleic acid of natural viral.

In another embodiment, embedded virus of the present invention is the reorganization hSARS virus that further comprises heterologous nucleotide sequence.According to the present invention, embedded virus can be by nucleotide sequence coded, wherein added heterologous nucleotide sequence or wherein endogenous or natural nucleus glycoside acid sequence is replaced by heterologous nucleotide sequence to genome.

According to the present invention, embedded virus is by the virus vector coding of the present invention that further comprises heterologous nucleotide sequence.According to the present invention, embedded virus is by comprising or not comprise the virus vector coding that is not the genomic nucleic acid of natural viral.According to the present invention, embedded virus is encoded by virus vector, wherein adds, inserts heterologous nucleotide sequence or replaced natural or the non-natural sequence.According to the present invention, but the embedded virus origin comes from the nucleotide sequence coded of the different strains of hSARS virus or variant.Specifically, embedded virus is by nucleotide sequence coded, the described nucleotide sequence coded different strains of hSARS virus or the antigenic peptide of variant of deriving from.

Embedded virus is useful especially (Tao etc., J.Virol.72,2955-2961 for producing anti-two or more viral recombiant vaccines; Durbin etc., 2000, J.Virol.74,6821-6831; Skiadopoulos etc., 1998, J.Virol.72,1762-1768; Teng etc., 2000, J.Virol.74,9317-9321).For example, it is contemplated that the virus vector that derives from one or more protein (vice versa) of hSARS virus, expression hSARS viral variants will protect the object of having inoculated this carrier to avoid the infection of natural hSARS virus and variant thereof.For purpose, can use attenuation and replication-defective virus as other virus with the living vaccine inoculation.(referring to the 6th page of PCT WO 02/057302 and the 23rd page, it is attached to herein by reference).

According to the present invention, the heterologous sequence of waiting to mix the virus vector of code book invention reorganization or embedded virus comprises from different strains or the variant of hSARS and obtaining or the deutero-sequence.

In certain embodiments, the chimeric or recombinant virus origin of the present invention comes from virus genomic virus vector coding, and wherein one or more sequences, intergenic region, end sequence or ORF are replaced by allos or non-natural sequence in whole or in part.In certain embodiments of the invention, embedded virus origin of the present invention comes from virus genomic virus vector coding, and wherein one or more heterologous sequences are inserted into or add in the carrier.

The selection of virus vector can be depending on virus infection to be treated or is protected from the kind of the object of virus infection.If human to liking, then available attenuation hSARS virus provides antigen sequence.

According to the present invention, can carry out virus vector artificial reconstructed, with provide hSARS virus, it is natural or the infection of artificial variant, analogue or derivative brings the antigen sequence of provide protection.Can carry out virus vector artificial reconstructed, so that a kind of, two kinds, three kinds or more kinds of antigen sequence to be provided.According to the present invention, antigen sequence can derive from different strains or variant or the different virus of same virus, same kind of viroid.

Expression product that obtains according to the present invention and/or reorganization or embedded virus particle can advantageously be applied in the vaccine product.Can carry out artificial reconstructedly to expression product of the present invention and embedded virus particle, to produce the vaccine of anti-multiple pathogenic agent, described pathogenic agent comprises virus and bacterial antigens, tumour antigen, allergen antigen and the autoantigen relevant with autoimmune disease.Specifically, can carry out artificial reconstructedly, can object of protection avoid the vaccine that hSARS virus or its natural or artificial variant, analogue or derivative infect to produce to embedded virus particle of the present invention.

In certain embodiments, can carry out artificial reconstructedly to expression product of the present invention and reorganization or embedded virus particle, to produce the vaccine of anti-multiple pathogenic agent, described pathogenic agent comprises virus antigen, tumour antigen and the autoantigen relevant with autoimmune disease.A method that realizes this target comprises modifies existing hSARS gene, makes to comprise exogenous array in described gene outer structure territory separately.At heterologous sequence is that these embedded viruses can be used to induce the protective immune response at the disease factor of these determinants of deriving under the epi-position or antigenic situation of pathogenic agent.

Therefore, the present invention relates to use virus vector and reorganization or embedded virus to prepare anti-multiple virus and/or antigenic vaccine.The present invention also comprises the recombinant virus that comprises virus vector, described virus vector derives from hSARS virus or its natural or artificial variant, analogue or derivative, contains the sequence of the phenotype (for example attenuation phenotype or enhanced antigenicity) that virus is had be more suitable for being used for vaccine product.Variation and modification can appear at coding region, intergenic region and leader sequence and the tailer sequence of virus.

The invention provides the host cell that comprises nucleic acid of the present invention or carrier.The plasmid vector or the virus vector that contain hSARS varial polymerases composition produce in prokaryotic cell prokaryocyte, to express described composition in relevant cell type (bacterium, insect cell, eukaryotic cell).The plasmid or the virus vector that contain hSARS genome total length copy or part copy produce in prokaryotic cell prokaryocyte, with at external or expression in vivo viral nucleic acid.Latter's carrier can contain other virus sequence to produce embedded virus or embedded virus albumen, can lack virus genomic some part with the generation replication-defective virus, and can contain sudden change, disappearance, displacement or insertion to produce attenuated virus.

The present invention also provides the host cell that comprises nucleic acid molecule of the present invention.In addition, the invention provides the host cell that infects hSARS virus, for example preserving number is hSARS virus or its natural or artificial variant, analogue or the derivative of CCTCC-V200303.In a specific embodiments, the present invention includes the continuous cell line of hSARS virus infection.Preferred this cell is a primate clone.These clones can cultivate and keep with known cell culture technology, and for example at Celis, Julio compiles, and 1994, Cell Biology LaboratoryHandbook, Academic Press is described in the N.Y..The various culture condition of these cells comprise culture medium prescription, oxygen, tension force, carbonic acid gas and minimizing serum level about concrete nutrition, can be selected and optimization by those skilled in the art.

Preferred cell of the present invention is an eukaryotic cell lines, preferred primate clone, and more preferably MC is, most preferably the tire RhMK is (for example FRhK-4), its of short duration or one or more total lengths of stably express or part hSARS albumen.This cell can be made by transfection (protein carrier or nucleic acid carrier), infection (virus vector) or transduction (virus vector), can be used for and described wild-type, attenuation type, replication defect type or mosaic type virus complementation.The clone of using among the present invention can be cloned with the known cell culture technology that those skilled in the art are familiar with.Can under the known conditions that is fit to cell proliferation, cultivate and the expansion cell with the commercial goods substratum from individual cells.

For example, it is freezing up to use that clone of the present invention keeps, and can heat under about 37 ℃ of temperature, adds the proper growth substratum then, for example contain 3% foetal calf serum (FBS) DMEM/F-12 (Life Technologies, Inc.).Described cell can be in moistening thermostat container about 5%CO ₂, hatch under 37 ℃ of temperature, up to fusion.For making described passage, remove growth medium, add 0.05% trypsinase and 0.53mM EDTA to cell.Separate described cell, cell suspension is collected in the centrifuge tube, centrifugal is cell precipitation.Can remove trypsin solution, cell precipitation is resuspended in the new growth medium.Then cell can be further in other growth bottle internal breeding to required density.

According to the present invention, continuous cell line comprises immortalized cell, and it was kept for 5,10,15,20,25 or 50 generations external at least.

The infectivity of hSARS (wild-type, attenuation type, replication defect type or mosaic type) copy can produce when the coexpression polysaccharase composition according to above-mentioned prior art.

In addition, can use of short duration or one or more total lengths of stably express or the proteinic eukaryotic cell of part hSARS.This cell can be made by transfection (protein carrier or nucleic acid carrier), infection (virus vector) or transduction (virus vector), can be used for and described wild-type, attenuation type, replication defect type or mosaic type virus complementation.

Virus vector of the present invention and embedded virus can be used for by stimulating humoral immunoresponse(HI), cellullar immunologic response or by stimulating antigenic tolerance and the immunity system of controlled plant.Object used herein refers to: people, primate, horse, ox, sheep, pig, goat, dog, cat, birds and rodent.

5.3. vaccine and antiviral agent

In a preferred embodiment, the invention provides protein molecule or hSARS virus-specific viral protein and function fragment thereof by nucleic acid encoding of the present invention.Useful protein molecule for example derives from and can comprise envelope protein (E albumen), integral protein (M albumen), spike protein (S albumen), nucleocapsid protein (N albumen), hemagglutinin esterase (HE albumen) and RNA RNA-dependent polysaccharase from the present invention's virus any gene of deutero-or genomic fragment.This molecule or its antigen fragment that this paper provided for example can be used in diagnostic method or the test kit, and are used for pharmaceutical composition such as subunit vaccine.Useful especially is by polypeptide or its antigen fragment shown in polypeptide, Figure 11 (SEQ ID NO:17-239,241-736 or 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964 or 1966-2470) of SEQ IDNO:1,11,13,15,2471,2472,2473,2474,2475 or 2476 nucleic acid sequence encodings, confession is sneaked into as antigen or subunit's immunogen, but also can use the totivirus of deactivation.What also be particularly useful is proteic substance by the genomic recombinant nucleic acid fragment coding of hSARS; more preferably in the preferred boundary of ORF, (, or be used to provide diagnostic antibody) especially in vivo or external (for example being used to produce the technology of synthetic antibody by display technique of bacteriophage or other) all causes the proteic substance of hSARS specific antibody or t cell response for example for protection purpose or therapeutic purpose.

5.3.1hSARS the attenuation of virus and variant thereof

Can carry out hSARS of the present invention virus or its variant genetic engineering modified, to demonstrate the attenuation phenotype.Specifically, virus of the present invention demonstrates the attenuation phenotype in the object that described virus gives as vaccine.Attenuation can be realized by any method that those of ordinary skill is known.Non-ly limited by theoretical, can be for example by using the virus that in predetermined host species, can not duplicate well in essence to produce the attenuation phenotype of virus of the present invention, for example with respect to the wild-type strain of virus, by reducing virus genomic duplicating, by the ability of reduction virus infection host cell, or by reducing the ability that virus protein is assembled into infectious virus particle.

In one embodiment, Bing Du infectivity is lowered 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2500 times, 2000 times, 1500 times, 1250 times, 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.Term used herein " infectivity " refers to the ability that virus enters, survives and duplicates in responsive host.In a specific embodiments, when in human host, growing, reduces if hSARS virus or the growth of its variant in human host are compared with non-attenuation hSARS virus or its variant, the infectivity of hSARS virus be known as reduction or weaken.The infectivity of virus can make the mensuration that ins all sorts of ways, and forms test, colorimetry, microscope and chemiluminescence such as but not limited to western blotting (protein), RNA trace (RNA), southern blotting technique (DNA), plaque.The infectivity of virus can be measured in zooblast, preferred primate cell, more preferably MC, the optimum cell of choosing.

In another embodiment, Bing Du replication is lowered 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2500 times, 2000 times, 1500 times, 1250 times, 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.Term used herein " replication " refers to the ability of virus replication, breeding and/or reproduction.Replication can use doubling time, multiple-copy rate, growth velocity and/or the half life of virus to measure.In a specific embodiments, when in human host, growing, reduces if hSARS virus or the growth of its variant in human host are compared with non-attenuation hSARS virus or its variant, the replication of hSARS virus be known as reduction or weaken.The replication of virus can make the mensuration that ins all sorts of ways, and forms test, colorimetry, microscope and chemiluminescence such as but not limited to western blotting (protein), RNA trace (RNA), southern blotting technique (DNA), plaque.In some cases, duplicate and transcribe synonym.The replication of virus can be measured in zooblast, preferred primate cell, more preferably MC, the optimum cell of choosing.

In another embodiment, Bing Du protein synthesis capacity is lowered 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2500 times, 2000 times, 1500 times, 1250 times, 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.Term used herein " protein synthesis capacity " refers to the ability of viral synthetic protein, and described protein is such as but not limited to envelope protein (E albumen), integral protein (M albumen), spike protein (S albumen), nucleocapsid protein (N albumen), hemagglutinin esterase (HE albumen) and RNA RNA-dependent polysaccharase.Protein synthesis capacity can and be measured and measure by protein synthesis rate (for example transcriptional level, translation skill) and the proteinic type of viral synthetic.In a specific embodiments, when in human host, growing, reduces if hSARS virus or the growth of its variant in human host are compared with non-attenuation hSARS virus or its variant, the protein synthesis capacity of hSARS virus be known as reduction or weaken.The protein synthesis capacity of virus can make the mensuration that ins all sorts of ways, and forms test, colorimetry, microscope and chemiluminescence such as but not limited to western blotting (protein), RNA trace (RNA), southern blotting technique (DNA), plaque.The protein synthesis capacity of virus can be measured in zooblast, preferred primate cell, more preferably MC, the optimum cell of choosing.

In another embodiment, Bing Du assemble ability is lowered 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2500 times, 2000 times, 1500 times, 1250 times, 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.Term used herein " assemble ability " refers to that virus assembling indispensable protein or albumen assembly become the ability of virion.In a specific embodiments, when in human host, growing, reduces if hSARS virus or the growth of its variant in human host are compared with non-attenuation hSARS virus or its variant, the assemble ability of hSARS virus be known as reduction or weaken.The assemble ability of virus can make the mensuration that ins all sorts of ways, and forms test, colorimetry, microscope and chemiluminescence such as but not limited to western blotting (protein), RNA trace (RNA), southern blotting technique (DNA), plaque.The assemble ability of virus can be measured in zooblast, preferred primate cell, more preferably MC, the optimum cell of choosing.

In another embodiment, Bing Du cytopathic effect is lowered 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2500 times, 2000 times, 1500 times, 1250 times, 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.Term used herein " cytopathic effect " refers to the infringement to infected host cell that infective virus causes.Virus infection can cause cellular abnormality (biochemical and morphologic) and/or necrocytosis (for example cracking).In a specific embodiments, when in human host, growing, reduces if hSARS virus or the growth of its variant in human host are compared with non-attenuation hSARS virus or its variant, the cytopathic effect of hSARS virus be known as reduction or weaken.The cytopathic effect of virus can make the mensuration that ins all sorts of ways, and forms test, colorimetry, microscope and chemiluminescence such as but not limited to western blotting (protein), RNA trace (RNA), southern blotting technique (DNA), plaque.The cytopathic effect of virus can be measured in zooblast, preferred primate cell, more preferably MC, the optimum cell of choosing.

Can carry out attenuation to virus of the present invention, so that one or more functional characters of virus are impaired.The attenuation phenotype of hSARS virus and variant thereof can detect by any method that those of ordinary skill is known.Candidate's virus can for example detect ability or its multiple-copy rate in cell culture system of its infection host.In certain embodiments, detect the attenuation phenotype of virus with the growth curve under the differing temps.For example, attenuated virus can be grown down at 35 ℃, but can not be 39 ℃ or 40 ℃ of growths down.In certain embodiments, available different clone is estimated the attenuation phenotype of virus.For example, attenuated virus can be grown in MC system, but can not grow in the human cell line, perhaps attenuated virus accessible virus titer difference in different clones.In certain embodiments, virus duplicating in the respiratory tract of meiofauna model (including but not limited to hamster, cotton mouse, mouse, cavy) is used to estimate the attenuation phenotype of virus.In other embodiments, the immunne response of virus induction includes but not limited to that antibody titers (for example testing by plaque reduction neutralization test or ELISA) is used to estimate the attenuation phenotype of virus.In a specific embodiments, plaque reduction neutralization test or ELISA carry out under low dosage.In certain embodiments, can detect hSARS virus causes pathological symptom in animal model ability.Virus causes that in animal model it is the indication of its attenuation phenotype that the ability of pathological symptom reduces.In a specific embodiments, detection candidate virus to the infection of nose, is produced as index with mucous in monkey model.

In some other embodiment, attenuation is to measure by making comparisons with the viral wild-type strain in attenuated virus source.In other embodiments, attenuation is to determine by comparing the growth of attenuated virus in different host systems.Therefore, as limiting examples, when growing in the human host, if the growth that hSARS virus or its variant are compared with non-attenuation hSARS or its variant in the human host reduces, then hSARS virus or its variant are called as attenuation.

In certain embodiments, attenuated virus of the present invention can infection host, perhaps can duplicate in the host, thereby produce infectious virus particle.But, to compare with the wild-type strain, the titre that the attenuated strain growth obtains is low, or grows slowlyer.Any method that available those of ordinary skill is known is determined the growth curve of attenuated virus and it is compared with the growth curve of wild-type virus.

In certain embodiments, attenuated virus of the present invention in people's cell, duplicate not as wild-type virus good.But attenuated virus is reproducible good in clone that lacks the Interferon, rabbit function such as Vero cell.

In other embodiments, attenuated virus of the present invention can infection host, can duplicate in the host, and the protein of virus of the present invention is embedded in the cytoplasmic membrane, but attenuated virus can not cause that the host produces new infectious virus particle.In certain embodiments, the attenuated virus infection host, in the host, duplicate and the efficient that causes virus protein to be embedded in host's the cytoplasmic membrane the same with wild-type hSARS.In other embodiments, its ability that causes virus protein to be embedded in the cytoplasmic membrane of host cell reduces attenuated virus with respect to wild-type virus.In certain embodiments, its ability of duplicating in the host reduces attenuation hSARS virus with respect to wild-type virus.Whether whether any technology that can use those of ordinary skill to know determines that virus whether can mammalian cell-infecting, can duplicate in the host and can cause virus protein to be embedded in host's the cytoplasmic membrane.

In certain embodiments, attenuated virus of the present invention can infection host.But opposite with wild-type hSARS virus is that attenuation hSARS virus can not be duplicated in the host.In a specific embodiments, attenuation hSARS virus energy infection host can cause the host that virus protein is embedded in its cytoplasmic membrane, but attenuated virus can not duplicate in the host.Whether any method that can use those of ordinary skill to know detects attenuation hSARS virus infection host and whether caused the host that virus protein is embedded in its cytoplasmic membrane.

In certain embodiments, the ability of attenuated virus infection host and the wild-type virus ability that infects identical host is compared and has been reduced.Any technology that can use those of ordinary skill to know determines whether virus can infection host.

In certain embodiments, sudden change (for example missense mutation) is introduced in the genome of virus, for example be introduced in SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences, have the virus of attenuation phenotype with generation.Sudden change (for example missense mutation) can be introduced in the structure gene and/or regulatory gene of hSARS virus.Sudden change can be to increase, replace, lack or their combination.Can screen the expectation function of this hSARS viral variants, as the infectivity in cell culture, replication, protein synthesis capacity, assemble ability and cytopathic effect.In a specific embodiments, missense mutation is a cold sensitive mutant.In another embodiment, missense mutation is the temperature-sensitive sudden change.In another embodiment, missense mutation prevents the normal process or the shearing of virus protein.

In other embodiments, disappearance is introduced in the genome of hSARS virus, causes the attenuation of virus.

In certain embodiments, Bing Du attenuation is to realize by the gene of replacing wild-type virus with the virogene of subgroup not of the same race, different or different variants.On the other hand, Bing Du attenuation is by realizing with deriving from different proteinic one or more ad hoc structures territories of planting the domain substitute wild-type virus of viral respective egg white matter.In some other embodiment, the attenuation of virus is realized by the proteinic one or more ad hoc structures of disappearance wild-type virus territory.

When attenuated vaccine is lived in use, also must consider its security.Described vaccine must not can cause disease.Any technology of vaccine safety that can make well known in the art all can be used in the present invention.Except that attenuation technology, also can use other technology.A limiting examples is to use the solubility heterologous gene that can not be incorporated in the virion film.For example, can use the shortage membrane spaning domain of single copy and the viral transmembrane protein soluble form in cytoplasmic structure territory.

Can use multiple test to detect the security of vaccine.For example, can use saccharose gradient test and neutralization test to detect security.The saccharose gradient test can be used to determine whether heterologous protein is inserted in the virion.If heterologous protein is inserted in the virion, then should detect virion causes symptom in appropriate animal model ability, because virus may obtain new, possible morbific character.

5.3.2 the preparation of vaccine

The invention provides the vaccine product that is used to prevent or treat the hSARS virus infection.In certain embodiments, vaccine of the present invention comprises the reorganization and the embedded virus of hSARS virus.In certain embodiments, virus is attenuation, deactivation or dead.

In the present invention's another embodiment in this respect, the inactivated vaccine goods can prepare by using routine techniques " to kill " embedded virus.On the disrupted meaning of its infectivity, inactivated vaccine is " dead ".It is desirable to, the infectivity of virus is destroyed, but does not influence its immunogenicity.For the preparation inactivated vaccine, embedded virus is grown in cell culture or in the allantois of chicken embryo, by the super centrifugal purification of district's band,, collect with formaldehyde or β-Bing Chunsuanneizhi deactivation.Obtained vaccine passes through intramuscular inoculation usually.

Inactivation of viruses can be replied with enhancing immunity with suitable adjuvant preparation.This adjuvant can include but not limited to inorganic gel, for example aluminium hydroxide; Surfactant such as lysolecithin, polyether glycol (pluronic polyol), polyanion; Peptide; Fat liquor; And have people's adjuvant such as the BCG and a spillikin bacillus (Corynebacterium parvum) of potential use.

Vaccine of the present invention can be multivalence or monovalent.The polyvalent vaccine preparation surpasses a kind of antigenic recombinant virus from expressing.

On the other hand, the present invention also provides the dna vaccination goods, wherein comprise nucleic acid or its fragment of hSARS virus (for example preserving number is the virus of CCTCC-V200303), or have the nucleic acid molecule of SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 sequences or its complement, analogue, derivative, fragment or part.In another embodiment, dna vaccination goods of the present invention comprise nucleic acid or its fragment of coding immunologic opsonin in conjunction with the antibody of hSARS virus.In the dna vaccination goods; dna vaccination comprises and has insertion segmental virus vector (for example deriving from hSARS virus, bacterial plasmid or other expression vector); described insertion fragment comprises the nucleic acid molecule of the present invention that operability ground links to each other with one or more controlling elementss, thereby feasiblely can be expressed in object of inoculation by the inoculation protein of described nucleic acid molecule encoding.This carrier can be prepared into reorganization or the chimeric vectors (referring to above 5.1 saving) that carries nucleic acid molecule of the present invention with recombinant DNA technology.

Described various for the allos carrier of DNA inoculation with anti-virus infection.For example, can be used to express the sequence of hSARS sequence rather than described virus or other pathogenic agent below with reference to the carrier of document description; Especially describe and be used for following carrier: hepatitis B virus (Michel, M.L. etc., 1995, DAN-mediated immunization to the hepatitis B surfaceantigen in mice:Aspects of the humoral response mimic hepatitis B viralinfection in humans, Proc.Natl.Aca Sci.USA 92:5307-5311; Davis, H.L. etc., 1993, DNA-based immunization induces continuous seretion ofhepatitis B surface antigen and high levels of circulating antibody, HumanMolec.Genetics 2:1847-1851), HIV virus (Wang, B. etc., 1993, Geneinoculation generates immune responses against human imunodeficiencyvirus type 1, Proc.Natl.Acad.Sci.USA 90:4156-4160; Lu, S. etc., 1996, Simian immunodeficiency viruus DNA vaccine trial in macques, J.Virol.70:3978-3991; Letvin, N.L. etc., 1997, Potent, protective anti-HIV immuneresponses generated by bimodal HIV envelope DNA plus proteinvaccination, Proc Natl Acad Sci USA.94 (17): 9378-83) and influenza virus (Robinson, HL etc., 1993, Protection against a lethal influenza viruschallenge by immunization with a haemagglutinin-exressing plasmidDNA, Vaccine 11:957-960; Ulmer, J.B. etc., Heterologous protectionagainst influenza by injection of DNA encoding a viral protein, Science259:1745-1749), and infectation of bacteria such as tuberculosis (Tascon, R.E. etc., 1996, Vaccination against tuberculosis by DNA injection, Nature Med.2:888-892; Huygen, K. etc., 1996, Immunogenicity and protective efficacyof a tuberculosis DNA vaccine, Nature Med. is 2:893-898) with parasitic infection such as malaria (Sedegah, M., 1994, Protection against malaria by immunizationwith plasmid DNA encoding circumsporozoite protein, Proc.Natl.Acad.Sci.USA 91:9866-9870; Doolan, D.L. etc., 1996, Circumventing geneticrestriction of protection against malaria with multigene DNAimmunization:CD8+T cell-interferon δ, and nitric oxide-dependentimmunity, J.Exper.Med., 1183:1739-1746).

Can use several different methods to import above-mentioned vaccine product.These methods include but not limited to mouth, intracutaneous, intramuscular, intraperitoneal, intravenously, the subcutaneous and interior approach of nose, and by scratch method (for example use and divide forked needle to scratch skin surface).

In addition, preferably import the embedded virus vaccine product at the natural infection approach of the pathogenic agent of its design by vaccine.Dna vaccination of the present invention can the salt brine solution form, by go in muscle or the skin to give (Wolff J.A. etc. with syringe and needle injection, 1990, Directgene transfer into mouse muscle in vivo, Science 247:1465-1468; Raz, E., 1994, Intradermal gene immunization:The possible role of DNAuptake in the induction of cellular immunity to viruses, Proc.Natl.Acd.Sci.USA 91:9519-9523).The mode that another kind gives dna vaccination is called " particle gun " method, by this method, the small gold bead that scribbles the target DNA molecule is launched into (Tang in the cell, D. etc., 1992, Genetic immunization is a simple method for elicitingan immune response, Nature 356:152-154).The comprehensive review of the method for relative dna vaccine is referring to Robinson, H.L., 1999, DNA vaccines:basic mechanismand immune responses (summary), Int.J.Mol.Med.4 (5): 549-555; Barber, B., 1997, Introduction:Emerging vaccine strategies, Seminars inImmunology 9 (5): 269-270; And Robinson, H.L. etc., 1997, DNA vaccines, Seminars in Immunology 9 (5): 271-283.

The patient who is given vaccine is preferably Mammals, most preferably is the people, but also can be the non-human animal, includes but not limited to ox, horse, sheep, pig, fowl (for example chicken), goat, cat, dog, hamster, mouse and rat.

5.3.3. adjuvant and carrier molecule

In certain embodiments, the hSARS related antigen is with one or more adjuvant administrations.In one embodiment, the hSARS related antigen is with inorganic salt adjuvant or the administration of inorganic salt gel adjuvant.This inorganic salt adjuvant and inorganic salt gel adjuvant include but not limited to aluminium hydroxide (ALHYDROGEL, REHYDRAGEL), phosphaljel, Adju-Phos (ADJU-PHOS) and calcium phosphate.

In another embodiment, the hSARS related antigen is with the administration of immunostimulation adjuvant.This class adjuvant includes but not limited to cytokine (interleukin-2 for example, interleukin-7, il-1 2, granulocyte-macrophage colony stimutaing factor (GM-CSF), interferon-, il-1 β (IL-1 β) and IL-1 β peptide or Sclavo peptide), the liposome of factor-containing, triterpenes glucosides or saponin(e (for example QuilA and QS-21, also at trade mark STIMULON, ISCOPREP sells down), Muramyl dipeptide (MDP) derivative such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (threonyl-MDP, under trade mark TERMURTIDE, sell), GMDP, N-acetyl-go first muramyl-L-alanyl-D-isoglutamine, N-acetyl muramyl-L-alanyl-D-isoglutamine acyl-L-L-Ala-2-(1 '-2 '-two palmityls-sn-glyceryl-3-hydroxyl phosphorus acyloxy) ethamine, MTP-PE (MTP-PE), unmethylated CpG dinucleotides and oligonucleotide such as DNA of bacteria and fragment thereof, LPS, one phosphinylidyne lipid A (3D-MLA sells under trade mark MPL) and polyphosphonitrile.

In another embodiment, used adjuvant is special adjuvant, include but not limited to emulsion, for example Freund's complete adjuvant, Freund's incomplete adjuvant, for example with the squalene of segmented copolymer such as L-121 (polyoxypropylene/polyoxyethylene is sold under trade mark PLURONIC L-121) preparation or squalane oil-in-water adjuvant formulation such as SAF and MF59, liposome, virosome, liposome volume (cochleate) and the immunostimulating complex under trade mark ISCOM, sold.

In another embodiment, use particle adjuvant.Polymkeric substance (poloxamer particle), soluble polymer (polyphosphonitrile) and virus-like particle (VLP) that particle adjuvant includes but not limited to the homopolymer (PGA) of the homopolymer (PLA) of biodegradable and biocompatibility polyester, lactic acid and oxyacetic acid and their multipolymer, rac-Lactide-glycolide copolymer (PLGA) particulate, can self-association become particulate be as the recombinant protein particulate, for example hepatitis B surface antigen (HbsAg).

Operable another class adjuvant comprises the mucous membrane adjuvant, include but not limited to from intestinal bacteria (Escherichia coli) heat-labile toxin (LT), from cholera holotoxin (CT) and choleratoxin B subunit, mutant strain toxin (for example LTK63 and LTR72), particulate and the polymerized liposome of vibrio cholerae (Vibriocholerae).

In other embodiments, the adjuvant of above-mentioned any kind can make up mutually or be used in combination with other adjuvant.For example, the limiting examples that can be used to give the combination adjuvant preparation of hSARS related antigen of the present invention comprises the liposome that comprises immunostimulation protein, cytokine, T cell and/or B cell peptide; Or have or not with the microorganism of embedding IL-2 or contain the particulate of enterotoxin.Other adjuvant well known in the art is also included within the scope of the present invention (referring to Vaccine Design:The Subunit and Adjuvant Appoach, the 7th chapter, MichaelF.Powell and Mark J.Newman (editor), Plenum Press, New York, 1995, its by reference integral body be attached to herein).

The effectiveness of adjuvant can be induced at the antibody of immunogenic polypeptide (containing the hSARS polypeptide epitope) by measurement and be determined that described antibody produces by give this peptide species in the vaccine that also comprises various adjuvants.

Described polypeptide can be mixed with the vaccine of neutral form or salt form.The acceptable salt of medicine comprises acid salt (free amine group by peptide forms) and the salt that forms with mineral acid (for example hydrochloric acid or phosphoric acid) or organic acid (for example acetate, oxalic acid, tartrate, toxilic acid etc.).The salt that forms by free carbonyl also can derive from mineral alkali, for example sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ironic hydroxide, and organic bases, for example Isopropylamine, Trimethylamine 99,2-ethylaminoethyl alcohol, Histidine, PROCAINE HCL, PHARMA GRADE etc.

5.4. the preparation of antibody

Antibody can separate from the serum of the object that infects SARS.Specific recognition polypeptide of the present invention, such as but not limited to comprising SEQ ID NO:2,12 or 14 polypeptide of sequence or the polypeptide shown in Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and Figure 12 (SEQ IDNO:1109-1589,1591-1964,1966-2470) or antibody or its Fab of hSARS epi-position, can be used to detect, screen and separate polypeptide of the present invention or its fragment similar sequence of other biological species of maybe may encoding like enzyme.For example, in a specific embodiments, immunologic opsonin can be used in the various vitro detection tests in conjunction with antibody or its fragment of hSARS epi-position, comprise enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, western blotting etc., to detect the polypeptide of polypeptide of the present invention or preferred hSARS virus in sample biological example material, described biomaterial comprises cell, cell culture medium (bacterial cell substratum for example, the mammalian cell substratum, the insect cell substratum, yeast cell substratum etc.), blood, serum, blood plasma, saliva, urine, ight soil, tissue, phlegm, nasopharynx aspirate etc.

Any epi-position of polypeptide of the present invention or hSARS virus is had specific antibody can be produced by any appropriate methodology well known in the art.The polyclonal antibody of anti-purpose antigen (for example preserving number is CCTCC-V200303 or the hSARS virus that comprises SEQ ID NO:15 nucleotide sequence) can be by produced in several ways well known in the art.For example, antigen can be given various host animals, include but not limited to rabbit, mouse, rat etc., produce the antiserum(antisera) that contains the antigen-specific polyclonal antibody to induce.Can use multiple adjuvant to come enhancing immunity to reply, depend on host species, adjuvant includes but not limited to Fu Shi (fully with incomplete) adjuvant, inorganic gel such as hydrogen aluminium, surfactant such as lysolecithin, polyether glycol, polyanion, peptide, fat liquor, keyhole Hemocyanin, dinitrophenol and the mankind are had adjuvant such as the BCG (bacille Calmette-Guerin vaccine) and the spillikin bacillus of potential use.This adjuvant is also known in this area.

Monoclonal antibody can be used multiple technology preparation well known in the art, comprises and uses hybridoma technology, recombinant chou technology and phage display techniques or their combination.For example monoclonal antibody can be used hybridoma technology production, comprise hybridoma technology well known in the art and for example at Harlow etc., Antibodies:A Laboratory Manual, (Cold Spring HarborLaboratory Press, the 2nd edition, 1988); Hammerling etc., Monoclonal Antibodiesand T-Cell Hybridomas, the hybridoma technology of lecturing in the 563-681 page or leaf (Elsevier, N.Y., 1981) (both integral body be attached to herein) by reference.Term used herein " monoclonal antibody " is not limited to the antibody by hybridoma technology production.Term " monoclonal antibody " refers to derive from single clone's antibody, comprises any eukaryotic cloning, protokaryon clone and phage clone, rather than refers to the method for antibody producing.

The method of using hybridoma technology production and screening antibody specific is conventional with well known in the art.In limiting examples, mouse can or be expressed this antigenic cellular immunization with purpose antigen.In case detect immunne response, for example in mice serum, detect antibody to antigen-specific, then gather in the crops mouse spleen, separating Morr. cell.By the technology of knowing splenocyte and any suitable myeloma cell are merged then.Select and the clone hybridization knurl by limiting dilution.Detect the cell of the antibody of secretion energy conjugated antigen among the hybridoma clone then with method well known in the art.Usually the ascites that contains high-level antibody can be by producing with positive hybridoma clone intraperitoneal inoculation mouse.

The antibody fragment of identification defined epitope can produce by known technology.For example, Fab and F (ab ') ₂Fragment can for example papoid (being used to produce the Fab fragment) or stomach en-(be used to produce F (ab ') by using enzyme ₂Fragment), immunoglobulin molecules being carried out protease hydrolysis produces.F (ab ') ₂Fragment contains variable region, CH1 district and the hinge area of whole light chains and heavy chain.

Antibody of the present invention or its fragment also can be produced by any antibody synthetic method well known in the art, specifically by chemical synthesis, or preferably produce by recombination and expression techniques.

The nucleotide sequence of encoding antibody can be from the retrievable any information acquisition of those of ordinary skills (promptly obtain or obtain by conventional clone and sequential analysis from Genbank, document).If containing the clone of the nucleic acid of coding specific antibodies or its epi-position binding fragment can not obtain, but the sequence of antibody molecule or its epi-position binding fragment is known, then encoding the nucleic acid of immunoglobulin (Ig) can be synthetic by chemical method, or obtain from suitable source (antibody cDNA library for example, or from cDNA library that any tissue of expressing antibodies or cell (as selecting to be used for the hybridoma of expressing antibodies) produce or from isolating nucleic acid wherein, preferred poly A+RNA), by use can with 3 of sequence ' and the synthetic primer of 5 ' terminal hybridization carry out pcr amplification, or specific gene sequence is had specificity by using, the oligonucleotide probe that for example is used for differentiating from the cDNA library of encoding antibody the cDNA clone is cloned and is obtained.The amplification of nucleic acid that produces by PCR can use any method well known in the art to be cloned in the reproducible cloning vector subsequently.

In case determined the nucleotide sequence of antibody, the nucleotide sequence of antibody can use the method for operation nucleotide sequence well known in the art, for example recombinant DNA technology, site-directed mutagenesis, PCR etc. are (referring to for example above-mentioned Sambrook etc. and volumes such as Ausubel, Current Protocolsin Molecular Biology, John Wiley﹠amp; Sons, NY, they by reference integral body be attached to herein) operate, with by for example the epi-position of antibody in conjunction with the territory in or can strengthen or reduce in the bioactive antibody any part of antibody and introducing aminoacid replacement, disappearance and/or insertion, produce antibody with different aminoacids sequence.

The recombinant expressed expression vector that needs to make up the nucleotide sequence that contains encoding antibody of antibody.In case obtain encoding antibody molecule or the heavy chain of antibody or the nucleotide sequence of light chain or its part, the carrier that is used to produce antibody molecule can pass through recombinant DNA technology, uses the technology well known in the art of each joint discussion of front to produce.The method that can use those of ordinary skills to know makes up and contains antibody coding sequence and the suitable expression vector of transcribing and translate control signal.These methods comprise for example interior genetic recombination of extracorporeal recombinant DNA technology, synthetic technology and body.The nucleotide sequence of one or more complementary determining regions (CDR) of the epi-position binding fragment of encoding heavy chain variable region, variable region of light chain, heavy chain and variable region of light chain, heavy chain and/or variable region of light chain or antibody can be cloned in this carrier and express.Then, so the expression vector of preparation can be introduced in the proper host cell with expressing antibodies.Therefore, the present invention includes the host cell that contains polynucleotide, described polynucleotide encoding has specific antibody to polypeptide of the present invention or its fragment.

Host cell can be with two kinds of expression vector cotransfections of the present invention, first kind of vector encoded heavy chain polypeptides derived, the polypeptide of second kind of vector encoded derived light chain.But described two kinds of carriers can contain identical selective marker so that the expression of heavy chain and light chain polypeptide equates, but or contain different selective markers, to guarantee to keep two kinds of plasmids.In addition, also can use coding and the single carrier that can express heavy chain polypeptide and light chain polypeptide.In this case, light chain should be positioned at the front of heavy chain, with avoid occurring excessive poisonous free heavy chain (Proudfoot, 1986, Nature, 322:52 and Kohler, 1980, Proc.Natl.Acad.Sci.USA, 77:2197).The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna.

In another embodiment, antibody also can use various phage rendering method well known in the art to produce.In the phage rendering method, the function antibody structural domain is present on the surface of the phage particle that carries its coded polynucleotide sequence.In a specific embodiments, this phage can be used to present from the antigen binding domain of all the components or combinatorial antibody library (for example the people's or mouse) expression, as Fab and Fv or the stable Fv of disulfide linkage.Expressing the phage of the antigenic antigen binding domain of binding purposes can select or identify with antigen, for example applying marking antigen or combined or capture the antigen of solid surface or bead.The phage of using in these methods is filobactivirus normally, comprises fd and M13.Antigen binding domain is expressed as and phage gene III or the proteinic recombinant fusion protein of gene VIII.The example that can be used to prepare immunoglobulin (Ig) of the present invention or its segmental phage rendering method is included in disclosed method in the following document: Brinkman etc., 1995, J.Immunol.Methods, 182:41-50; Ames etc., 1995, J.Immunol.Methods, 184:177-186; Kettleborough etc., 1994, Eur.J.Immunol., 24:952-958; Persic etc., 1997, Gene, 187:9-18; Burton etc., 1994, Advances in Immunology, 57:191-280; PCT application number PCT/GB91/01134; PCT publication number WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; And U.S. Patent number 5,698,426,5,223,409,5,403,484,5,580,717,5,427,908,5,750,753,5,821,047,5,571,698,5,427,908,5,516,637,5,780,225,5,658,727,5,733,743 and 5,969,108; More than each document by reference integral body be attached to herein.

As described in above reference, after carrying out the phage selection, separable and use antibody coding region from phage to produce all antibody, the fragment that comprises people's antibody or any other needs, and can be expressed among the host of any needs, comprise mammalian cell, insect cell, vegetable cell, yeast and bacterium, for example the following detailed description of.For example, also can use recombinant production Fab, Fab ' and F (ab ') ₂Segmental technology is used for example disclosed method well known in the art in following document: PCT publication number WO 92/22324; Mullinax etc., 1992, BioTechniques, 12 (6): 864-869; And Sawai etc., AJRI, 34:26-34,1995; And Better etc., 1988, Science, 240:1041-1043 (above each document integral body by reference is attached to herein).The example that can be used to produce the technology of strand Fv and antibody is included in the technology of describing in the following document: U.S. Patent number 4,946,778 and 5,258,498; Huston etc., 1991, Methods in Enzymology, 203:46-88; Shu etc., 1993, PNAS, 90:7995-7999; And Skerra etc., 1988, Science, 240:1038-1040.

In case antibody molecule of the present invention is produced by above-mentioned any method, it can carry out purifying by the method for any purifying immunoglobulin molecules well known in the art subsequently, described method is chromatography (ion-exchange chromatography for example for example, affinity chromatography, especially carry out behind a-protein and the protein G purifying affinity chromatography, and the exclusion column chromatography to specific antigen), the standard technique of centrifuging, differential solubility method or any other protein purification.In addition, antibody of the present invention or its fragment can merge with allogeneic polypeptide sequence as herein described or well known in the art, to promote purifying.

For some application, be included in and use antibody and vitro detection test in the human body, preferably use chimeric antibody, humanized antibody or people's antibody.Chimeric antibody is the antibody molecule that the different piece of wherein antibody derives from different animal species, for example has variable region that derives from mouse monoclonal antibody and the antibody that derives from the constant region of human normal immunoglobulin.The method of producing chimeric antibody is well known in the art.Referring to for example Morrison, 1985, Science, 229:1202; Oi etc., 1986, BioTechniques, 4:214; Gillies etc., 1989, J.Immunol.Methods, 125:191-202; U.S. Patent number 5,807,715,4,816,567 and 4,816,397, more than each document by reference integral body be attached to herein.Humanized antibody be from the non-human species can be in conjunction with required antigenic antibody molecule, it has one or more complementary determining regions from the non-human species (CDR) and from the framework region of human normal immunoglobulin molecule.Usually, the framework residue in people's framework region can be replaced by the corresponding residue from the CDR donor antibody, with change, the antigenic combination of advantageous embodiment.These frameworks replace and can identify by method well known in the art, for example by modeling is carried out in the interaction of CDR and framework residue, identifying antigen in conjunction with important framework residue, and by sequence relatively, to identify unusual framework residue at specific position.Referring to for example Queen etc., U.S. Patent number 5,585,089; Riechmann etc., 1988, Nature, 332:323, above document integral body by reference is attached to herein.Antibody can use multiple various technology well known in the art to carry out humanization, and (EP 239,400 to comprise for example CDR grafting; PCT publication number WO 91/09967; U.S. Patent number 5,225,539; 5,530,101 and 5,585,089), (EP 592,106 for surface gummed (veneering) or resurfacing (resurfacing); EP 519,596; Padlan, 1991, Molecular Immunology, 28 (4/5): 489-498; Studnicka etc., 1994, ProteinEngineering, 7 (6): 805-814; Roguska etc., 1994, Proc Natl.Acad.Sci.USA, 91:969-973) and chain reorganization (U.S. Patent number 5,565,332), more than each document by reference integral body be attached to herein.

Fully human antibodies is particularly suitable for people patient is treated processing.People's antibody can prepare by multiple method well known in the art, comprises using the antibody library that derives from the human normal immunoglobulin sequence to prepare by above-mentioned phage display techniques.Referring to U.S. Patent number 4,444,887 and 4,716,111; And PCT publication number WO 98/46645; WO98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735 and WO 91/10741, more than each document by reference integral body be attached to herein.

People's antibody also can use the transgenic mice of can not the endogenous immunoglobulin (Ig) of expressive function but can the expressing human immunoglobulin gene to produce.The summary of the technology of relevant this production people antibody is referring to Lonberg and Huszar, 1995, Int.Rev.Immunol., 13:65-93.About the technology of this production people's antibody and human monoclonal antibodies and the going through of scheme of producing this antibody, referring to for example PCT publication number WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European patent number 0598877; U.S. Patent number 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771 and 5,939,598, more than each document by reference integral body be attached to herein.In addition, can with for example Abgenix, Inc. (Fremont, CA), Medarex (NJ) and Genpharm (San Jose, company's agreement such as CA), with provide use that similar above-mentioned technology produces at selected antigenic people's antibody.

The fully human antibodies of the selected epi-position of identification can use the technology that is called " guiding is selected " to produce.In this method, selected non-human monoclonal antibodies such as mouse antibodies are used to instruct the fully human antibodies of selecting the same epi-position of identification.(Jespers etc., 1988, Bio/technology, 12:899-903).

The antibody that merges with heterologous polypeptide or put together can be used in external immunity test well known in the art or the purification process (for example affinity chromatography).Referring to for example PCT publication number WO93/21232; EP 439,095; Naramura etc., 1994, Immunol.Lett., 39:91-99; U.S. Patent number 5,474,981; Gillies etc., 1992, PNAS, 89:1428-1432 and Fell etc., 1991, J.Immunol., 146:2446-2452, more than each document by reference integral body be attached to herein.

Antibody is also adsorbable to solid support, and this is useful especially to immunity test and purifying polypeptide of the present invention or its fragment, derivative, analogue or variant or the similar molecule that has with the similar enzymic activity of polypeptide of the present invention.This solid support includes but not limited to glass, Mierocrystalline cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

5.5 pharmaceutical composition and medicine box

The present invention includes the pharmaceutical composition that comprises antiviral agent of the present invention.In a specific embodiments, antiviral agent be the immunologic opsonin combination and in and the antibody of hSARS virus or its natural or artificial variant, analogue or derivative or its any derived protein.In the described virucidin and the infectivity of virus, and the resist the disease that when giving animal with wild-type virus subsequently, watches for animals.

In another embodiment, described antiviral agent is polypeptide of the present invention or nucleic acid molecule.Pharmaceutical composition has the effectiveness of preventative antiviral agent, can give to be exposed to or to have expected the object that is exposed to virus.

Known have various drug delivery systems to can be used to give pharmaceutical composition of the present invention, described drug delivery system for example liposomes encloseization, particulate, microcapsule, the reconstitution cell that can express mutated viruses, receptor mediated endocytosis effect (referring to for example Wu and Wu, 1987, J.Biol.Chem.262:44294432).The method of introducing includes but not limited in intracutaneous, intramuscular, intraperitoneal, intravenously, subcutaneous, the nose, epidural, cut and oral route.Compound can pass through any administration easily, for example by inculcating or large bolus injection, absorbing by epithelium or mucous membrane skin inner layer (for example oral mucosa, rectal mucosa and intestinal mucosa etc.), and also can be with other biologically active agent administration.Administration can be whole body administration or topical.In preferred embodiments, expectation is introduced lung by any suitable way with pharmaceutical composition of the present invention.Also can prepare with the aerosolization agent and implement pulmonary administration for example by using sucker or atomizer.

In a specific embodiments, the zone that expectation need be treated pharmaceutical composition topical administration of the present invention; This for example (without limitation) by the part in the operation inculcate, topical application (for example with postoperative wound dressings), by injection, by conduit, realize by suppository or by implant, described implant is porous, atresia or gel-like material, comprises film such as silicon rubber (sialastic) film or fiber.In one embodiment, can be by coming administration at the position (or from front) of infected tissue direct injection.

In another embodiment, pharmaceutical composition can in vesicle, especially liposome, send and pass (referring to Langer, 1990, Science 249:1527-1533; Treat etc., Liposomesin the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (editor), Liss, New York, 53-365 page or leaf (1989); Lopez-Berestein, ibid, the 317-327 page or leaf; Usually referring to ibid).

In another embodiment, pharmaceutical composition can controlled release system send to be passed.In one embodiment, (referring to Langer, ibid can to use pump; Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:201; Buchwald etc., 1980, Surgery 88:507; With Saudek etc., 1989, N.Engl.J.Med.321:574).In another embodiment, can use polymer materials (referring to Medical Applications of Controlled Release, Langer and Wise (editor), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (editor), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol.Sci.Rev.Macromol.Chem.23:61; Also referring to Levy etc., 1985, Science 228:190; During etc., 1989, Ann.Neturol.25:351; Howard etc., 1989, J.Neurosurg.71:105).In another embodiment, controlled release system can place the composition target be lung near, thereby a part that only needs body dose gets final product (referring to for example Goodson, Medical Applications of Controlled Release, ibid, the 2nd volume, 115-138 page or leaf (1984)).

Other controlled release system has discussion in the summary (1990, Science 249:1527-1533) of Langer.

Pharmaceutical composition of the present invention comprises the work for the treatment of significant quantity but attenuation, deactivation or dead hSARS virus, or reorganization or chimeric hSARS virus and medicine acceptable carrier.In a specific embodiments, term " medicine is acceptable " refers to obtain authority's approval of federal government or state government, or is put in American Pharmacopeia or other pharmacopeia of generally acknowledging usually, in animal, more specifically use in the mankind.Term " carrier " refers to the thinner, adjuvant, vehicle or the medium that give with pharmaceutical composition.This pharmaceutical carrier can be sterile liquid Ru Shui and oil, comprises the liquid in oil, animal, plant or synthetic source, as peanut oil, soybean oil, mineral oil, sesame wet goods.When intravenously gave pharmaceutical composition, water was preferred carrier.Salt brine solution and glucose and aqueous glycerin solution also can be used as liquid vehicle, in particular for Injectable solution.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Wingdale, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium-chlor, skim-milk, glycerine, propylene, ethylene glycol, water, ethanol etc.As needs, composition also can contain a spot of wetting agent or emulsifying agent or pH buffer reagent.Forms such as these compositions can solution, suspensoid, emulsion, tablet, pill, capsule, powder, sustained release dosage occur.Composition can be mixed with suppository with traditional tackiness agent and carrier such as triglyceride level.Oral dosage form can comprise the carrier of standard, as pharmaceutical grade N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc.The example of suitable pharmaceutical carrier has description in " Remington ' s Pharmaceutical Sciences " of E.W.Martin work.Formulation should adapt with administering mode.

In preferred embodiments, composition is mixed with according to conventional procedure and is fit to give human pharmaceutical composition for intravenously.Be typically, supplying the composition of intravenous administration is the solution of solutions in sterile isotonic aqueous buffer.In case of necessity, composition also can comprise solubilizing agent and local anesthetic such as lignocaine, to alleviate the pain of injection site.Usually, each composition for example as the lyophilized powder in sealed vessel such as ampoule or pouch or there is not aqueous concentrate, is indicated the amount of active medicine with unit dosage form separately or mixed supply on the container.Under the situation of composition by the transfusion administration, it is available to be equipped with aseptic pharmaceutical grade water or the brinish infusion bottle is adjusted.By under the situation of drug administration by injection, can provide an ampoule Injectable sterile water or a salt solution at composition, so that before administration, mix each composition.

Pharmaceutical composition of the present invention can be mixed with neutral form or salt form.The acceptable salt of medicine comprises the salt that forms with free amine group, as derive from the salt of hydrochloric acid, phosphoric acid, acetate, oxalic acid, tartrate etc., and the salt that forms with free carboxy, as derive from the salt of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ironic hydroxide, Isopropylamine, triethylamine, 2-ethylaminoethyl alcohol, Histidine, PROCAINE HCL, PHARMA GRADE etc.

The amount that pharmaceutical composition of the present invention can effectively be treated concrete symptom or illness must depend on the character of symptom or illness, can determine by the clinical technology of standard.In addition, can choose the dosage range that adopts in vitro tests to help determine the best wantonly.Exact dosage desired to be adopted also must depend on the severity of route of administration and disease or symptom in preparation, and should decide according to the judgement of practitioner and each patient's situation.But the suitable dose scope of intravenous administration is the about 20-500 microgram of per kilogram of body weight active compound normally.The normally about 0.01pg/kg body weight of the suitable dose scope of intranasal administration is to the 1mg/kg body weight.Effective dose can be known by inference from deriving from amount effect curve external or the animal model test macro.

Suppository contains the activeconstituents of 0.5%-10% (weight) usually; Oral dosage form preferably contains the activeconstituents of 10%-95%.

The present invention also provides medicine parcel or the medicine box that comprises one or more containers, and one or more pharmaceutical composition compositions of the present invention are housed in the container.Can choose what follow this container wantonly is the letter of information of prescribed form of government bodies of production, use or the sale of a management medicine or biological products, and this letter of information has reflected the approval of government bodies to producing, using or sell human drugs or biological products.In a preferred embodiment, medicine box comprises antiviral agent of the present invention, for example to SEQ ID NO:1,11,13,15,2471,2472,2473,2474, the polypeptide of 2475 or 2476 nucleic acid sequence encodings, or Figure 11 (SEQ ID NO:17-239,241-736 or 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964 or 1966-2470) shown in polypeptide, or any hSARS epi-position, or polypeptide of the present invention or protein, or nucleic acid molecule of the present invention has specific antibody, but its Individual existence, or and adjuvant, antiviral, microbiotic, pain killer, bronchodilator or other medicines can be accepted excipient composition.

The present invention further comprises this medicine box, and it comprises container and the working instructions that contain pharmaceutical composition of the present invention.

5.6 detect test

The invention provides in and detect the method for immunologic opsonin in conjunction with the antibody of hSARS virus from SARS patient's biological sample such as cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm, nasopharynx aspirate etc.In a specific embodiments, described method comprises sample and the hSARS virus that directly is immobilized on the substrate, for example preserving number is the hSARS virus of CCTCC-V200303 or the hSARS virus contact with SEQ ID NO:15 genomic nucleic acid sequence, directly detects or pass through the allos anti-allotypic antibody indirect detection and the viral bonded antibody of mark then.In another embodiment, sample with by hSARS virus, for example preserving number is the hSARS virus of CCTCC-V200303 or the host cell contact with hSARS virus infection of SEQ IDNO:15 genomic nucleic acid sequence, and combined then antibody can detect by the immunofluorescent test that following 6.5 joints are described.

The illustrative methods that whether polypeptide of the present invention or nucleic acid exist in the detection of biological sample comprises, obtain biological sample from various sources, sample is contacted the existence of hSARS virus in the test sample like this with the compound or the reagent of epi-position that can detect hSARS virus or nucleic acid (for example mRNA, geneome RNA).The preferred reagent that detects hSARS mRNA of the present invention or geneome RNA be can with the labeling nucleic acid probe of the mRNA of code book invention polypeptide or geneome RNA hybridization.This nucleic acid probe can be the nucleic acid molecule that for example comprises or be made up of SEQ ID NO:1,11,13,15,16,240,737,1108,1590,1965,2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its complement, analogue, derivative, fragment or part, as length is at least 15,20,25,30,50,100,250,500,750,1000 or the oligonucleotide of more continuous nucleotides, and it is enough to specificity and hSARS mRNA or geneome RNA hybridization under stringent condition.

In another preferred specific embodiments, the existence of hSARS virus is to use following primer to detect by RT-polymerase chain reaction (RT-PCR) in the sample, described primer makes up based on the genomic partial nucleotide sequence of hSARS virus (for example hSARS virus of preservation searching number CCTCC-V200303), or makes up based on SEQ ID NO:1,11,13,15,16,240,737,1108,1590 or 1965 nucleotide sequences.In a non-limiting specific embodiments, the preferred primer that is used for the RT-PCR method is: 5 '-TACACACCTCAGCGTTG-3 ' (SEQ ID NO:3) and 5 '-CACGAACGTGACGAAT-3 ' (SEQ ID NO:4), at 2.5mM MgCl ₂Exist down, thermal cycling such as but not limited to 94 ℃ 8 minutes, 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ circulations of 1 minute 40 times (6.7 joints in addition vide infra) subsequently.In preferred specific embodiments, the invention provides real-time quantitative PCR and test existing: from the sample extracted total RNA with hSARS virus in the following detection of biological sample, extracted total RNA is carried out reverse transcription obtains cDNA, use specific primer as primer with SEQ ID NO:3 and 4 nucleotide sequences and fluorescence dye as Green I (it can send fluorescence when non-specific when combining with double-stranded DNA) makes cDNA experience PCR reaction.In another preferred specific embodiments, the real-time quantitative PCR that uses among the present invention is Test (referring to above 5 saving).In particular, for detect the real-time quantitative PCR test that hSARS virus exists in biological sample, the primer of use is preferably the primer with SEQ ID NO:2471 and 2472 nucleotide sequences.In this case, by Probe in detecting amplified production, preferred described probe have SEQ ID NO:2473 nucleotide sequence.

The another kind of preferred primer that uses in the test is the primer with SEQ ID NO:2474 and 2475 nucleotide sequences, and is preferred Probe has SEQ ID NO:2476 nucleotide sequence.Owing to after a series of thermal cyclings, produce the PCR product, extend the fluorescent signal of catching these reactions when step finishes at each, thus can be based on the viral load amount in the amplification figure quantitative assay sample (6.7 and 6.8 joints vide infra).

In another preferred specific embodiments, the existence of hSARS virus is measured with fluorescence cDNA microarray technology in the sample.The cDNA probe of a series of hSARS of deriving from viruses (for example preserving number CCTCC-V200303 or have the virus of SEQ ID NO:15 genomic nucleic acid sequence) is by reverse transcription and use the amplification preparation of suitable primer, and described primer makes up based on for example virus genomic partial nucleotide sequence of described hSARS or SEQ ID NO:1,11,13,15,16,240,737,1108,1590 or 1965 nucleotide sequences.This amplified production of purifying, and being fixed then is at chip for example on the sheet glass of poly-L-Lysine bag quilt, as the cDNA microarray.Extract total RNA from biological sample, reverse transcription in the presence of fluorescently-labeled Nucleotide.Represent that then the mark cDNA of mRNA in the sample contacts with immobilization cDNA probe on the microarray, detect and quantitatively in conjunction with the fluorescent signal of cDNA.Various dna microarray methods have been described, for example in Nucleic Acid Res.28 (22): 4552-7 (Kane, M.D. etc., 2000); Science 2000Sep 8; 289 (5485): 1757-60 (Taton, T.A. etc., 2000); Nature, 405 (6788): among the 827-836 (Lockhart, D.J. etc., 2000).

The another kind of reagent of preferred detection hSARS virus is the antibody of specificity in conjunction with polypeptide of the present invention or any hSARS epi-position, preferably has the antibody of detectable label.Described antibody can be polyclonal antibody, or monoclonal antibody more preferably.Can use complete antibody or its fragment (for example Fab or F (ab ') ₂).

The term " mark " that relates to probe or antibody comprise by with detectable substance coupling (being physical connection) to probe or antibody so that probe or antibody are carried out direct mark, and by with by another reagent react of direct mark so that probe or antibody are carried out indirect mark.The example of indirect labelling comprises with fluorescently-labeled second antibody and detects first antibody and with vitamin H dna probe is carried out end mark, detects with regard to available fluorescently-labeled streptavidin like this.Detection method of the present invention can be used to mRNA, protein (or any epi-position) or the geneome RNA in the test sample in external or the body.For example the technology of vitro detection mRNA comprises Northern hybridization, in situ hybridization, RT-PCR and RNA enzyme protection.The technology of vitro detection hSARS virus epitopes comprises enzyme linked immunosorbent assay (ELISA), western blotting, immuno-precipitation and immunofluorescence technique.The technology of vitro detection geneome RNA comprises Northern hybridization, RT-PCR and RNA enzyme protection.In addition, the technology of detection hSARS virus comprises the traget antibody of introducing at polypeptide in the body in the object organisms body.For example, antibody can be used radioactive mark's mark, and described radioactive mark can detect by standard imaging technique (comprising radioautography) existence and the position in the object organisms body.

In a specific embodiments, the inventive method further comprises from contrast object acquisition control sample, control sample is contacted with the compound or the reagent that can detect hSARS virus (mRNA or the geneome RNA of polypeptide for example of the present invention or code book invention polypeptide), thereby the existence of the mRNA of hSARS virus or polypeptide or coded polypeptide or geneome RNA in the test sample, and hSARS virus in the existence of the mRNA of hSARS virus or polypeptide or coded polypeptide in the control sample or geneome RNA and the testing sample or the mRNA of polypeptide or coded polypeptide or the existence of geneome RNA compared.

In a specific embodiments, the invention provides the diagnostic kit that comprises nucleic acid molecule, described nucleic acid molecule is suitable for detecting hSARS virus or its natural or artificial variant, analogue or derivative.In a specific embodiments, nucleic acid molecule has SEQ IDNO:2471 and 2472 nucleotide sequences.In specific embodiments, nucleic acid molecule has SEQID NO:2473 nucleotide sequence.In another embodiment, nucleic acid molecule has SEQID NO:2474 and 2475 nucleotide sequences.In specific embodiments, nucleic acid molecule has SEQ ID NO:2476 nucleotide sequence.

The present invention also comprises the test kit of the existence that detects hSARS virus of the present invention in the testing sample or polypeptide or nucleic acid.Described test kit can for example comprise the tagged compound or the reagent that can detect hSARS virus in the testing sample or polypeptide or nucleic acid encoding molecule, in certain embodiments, also comprise the amount of determining polypeptide in the sample or mRNA instrument (for example in conjunction with the antibody of polypeptide or with the DNA or the mRNA bonded oligonucleotide probe of coded polypeptide).Test kit also can comprise working instructions.

For the test kit based on antibody, it for example can comprise: (1) first antibody (for example being connected on the solid support), and it is in conjunction with polypeptide of the present invention or hSARS virus epitopes; Optional (2) different second antibody, it is in conjunction with polypeptide or first antibody, but and is conjugated on the detection reagent.

For test kit based on oligonucleotide, it for example can comprise: (1) oligonucleotide, detectable label oligonucleotide for example, its can with the nucleotide sequence of code book invention polypeptide or the sequence hybridization in the hSARS genome, or (2) a pair of primer, it can be used for increasing and contains the nucleic acid molecule of hSARS sequence.Described test kit also can comprise for example buffer reagent, sanitas or protein stabilizing agent.Described test kit also can comprise and be used to detect the composition (for example enzyme or substrate) that detectable reagent place needs.Described test kit also can contain control sample or a series of control sample, described control sample can be used to test and with contained the comparing of testing sample.Each composition of test kit is enclosed in the independent container usually, and all various containers are contained in the unitary package thing with working instructions.

5.7. shaker test

The invention provides the method that is used to identify the compound that suppresses hSARS virus infection host or host cell ability.In certain embodiments, the invention provides and be used for identifying and reduce the method for hSARS virus at the compound of host or host cell replication.Can use any technology screening that those of ordinary skill knows to destroy or reduce hSARS virus infection host and/or the compound of the ability of in host or host cell, duplicating.

In certain embodiments, the invention provides and be used for identifying and suppress the method for hSARS virus at the compound of Mammals or mammalian cell replication.More particularly, the invention provides the method that is used to identify the compound that suppresses hSARS virus infection Mammals or mammalian cell ability.In certain embodiments, the invention provides and be used for identifying and suppress the method for hSARS virus at the compound of mammalian cell replication.In a specific embodiments, mammalian cell is people's cell.

In another embodiment, cell is contacted and uses the hSARS virus infection with test compounds.In certain embodiments, control cultures is used the hSARS virus infection in test compounds in the presence of not.Cell can with before the hSARS virus infection, simultaneously or contact with test compounds afterwards.In a specific embodiments, cell is a mammalian cell.At one even more specifically in the embodiment, cell is people's cell.In certain embodiments, cell was hatched 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours or 1 day with test compounds at least.The titre of virus can be measured in any time of test.In certain embodiments, measure the time course of viral growth in the culture.If viral growth is suppressed or reduces in the presence of test compounds, then test compounds can be accredited as suppressing or reducing the growth of hSARS virus or infect effective.In a specific embodiments, test suppresses or the compound of reduction hSARS viral growth suppresses or reduce the ability of other viral growth speed, and/or tests its specificity to hSARS virus.

In one embodiment, give animal pattern with test compounds, the latter uses the hSARS virus infection.In certain embodiments, the contrast animal pattern is with the hSARS virus infection but do not give test compounds.Test compounds can be before with the hSARS virus infection, simultaneously or give afterwards.In a specific embodiments, animal pattern is a Mammals.At one even more specifically in the embodiment, animal pattern is to be but to be not limited to cotton mouse, mouse or monkey.Virus titer in the animal pattern can be measured in any time of test.In certain embodiments, measure the time course of viral growth in the culture.If viral growth is suppressed or reduces in the presence of test compounds, then test compounds can be accredited as suppressing or reducing the growth of hSARS virus or infect effective.In a specific embodiments, the compound of hSARS viral growth suppresses or reduces the ability of other viral growth speed in test inhibition or the reduction animal pattern, to test its specificity to hSARS virus.

6. embodiment

Following examples have illustrated the separation and the evaluation of novel hSARS virus.These embodiment should not be interpreted as limitation of the present invention.

Method and result

Use the Manual of ClinicalVirology of Wiedbrauk DL and Johnston SLG, Raven Press, New York, 1993 as general reference.

6.1 clinical subjects

This research comprises that the patient who defines revises in totally 50 The World Health Organization (WHO)s that meet SARS, they on February 26th, 2003 to being accepted for medical treatment (WHO.Severe acute respiratorysyndrome (SARS) 2000, Weekly Epidemiol Rec 78:81-83) by two acute diseases of the Hong Kong Special Administrative Region (HKSAR) ground district hospitals between March 26.This research also comprises lung's examination of living tissue of an other patient, and this patient suffers from typical SARS and accepted for medical treatment by the 3rd tame hospital.Briefly, the case definition of SARS is: (i) heating is 38 ℃ or higher; (ii) cough or breathe hard; (iii) the chest x-ray photo shows new lung's infiltration; And (iv) have and SARS patient's contact history or reactionless the experience antibiotic medicine covering (beta-lactam and macrolide medicine, fluoroquinolone or tsiklomitsin) of typical case and atypical pneumonia.

Collect nasopharynx aspirate and serum sample from all patients.Can obtain acute phase and convalescent serum and ight soil simultaneously on one's body from some patients.Lung's living tissue from a patient is handled, for carrying out virus culture, RT-PCR, conventional organization pathological examination and submicroscopy.Nasopharynx aspirate, ight soil and serum for the microbiological test that carries out other disease add under the blind test situation in this research, in contrast thing.

By attending doctor and Clinical microorganism scholar medical records is looked back and to be checked.Carry out conventional hematology, biological chemistry and microbiological examination, comprise microbial culture, the serological research of blood and phlegm, and collect the nasopharynx aspirate and carry out the virusology test.

6.2 clone

FRhK-4 (tire RhMK) cell is kept in containing the minimum essential medium (MEM) of 1% foetal calf serum, 1% Streptomycin sulphate and penicillin, 0.2% nystatin and 0.05% gentamicin sulphate.

6.3 virus infection

Use from two patients (" result " joint vide infra), be in 200 μ l clinical (nasopharynx aspirate) the sample infection FRhk-4 cell in the virus transportation substratum.With inoculating cell 37 ℃ of following incubations 1 hour.To contain the tryptic 1ml MEM of 1 μ g then and join in the culture, with cells infected incubation in 37 ℃ of incubators of 5% carbonic acid gas are provided.After incubation 2-4 days, observe the cytopathic effect that occurs in the cells infected.Cells infected is gone down to posterity become new FRhK-4 cell, observe cytopathic effect in back 1 day in inoculation.By influenza virus A, influenza virus B, respiratory syncytial virus, Parainfluenza type 1 virus, 2 types and 3 types, adenovirus and the human stroma lung virus (hMPV) of immunofluorescent test test cells infected, the test-results of all cases is all negative.Also by the influenza virus A and the human stroma lung virus of RP-PCR test cells infected, the result is negative.

6.4 morphology of virus is learned

Collect the as above cells infected of preparation, be centrifuged into granular substance, handle cell granulations shape thing, for carrying out the inspection of thin section transmission electron microscope.In the cell that infects two clinical samples, identify virion, but in not by the control cells of virus infection, do not have.From the isolating virion of cells infected 70-100 nanometer (Fig. 2) is arranged approximately.Viral capsid mainly is found in the vesicle of golgi body and endoplasmic reticulum, and discovery is also arranged in tenuigenin.In cytolemma, also find virion.

A viral isolates is carried out super centrifugal, gained cell granulations shape thing is carried out negative staining with phospho-wolframic acid.Shown virion like this with coronaviridae feature.Can't cause similar disease because the human coronary virus who generally acknowledges so far is known, the inventor thinks that described viral isolates has been represented and infects human new virus.

6.5 antibody response

For confirming that further this new virus causes SARS in infected patient, obtain serum sample on one's body from SARS patient, carry out neutralization test.With the serum (x50, x200, x800 and x1600) of typical case dilution with the FRhK-4 cell of the infection hSARS virus of acetone fixed 37 ℃ of following incubations 45 minutes.Use phosphate-buffered salt water washing incubation cell then, put together antibody staining with anti-human IgG-FITC.Washed cell is checked under fluorescent microscope then.In these experiments, in 8 SARS patients, find positive signal (Fig. 3), show that these patients have the IgG antibody response to this coronaviridae Novel Human Respirovirus.Contrast does not have signal to detect in 4 parts of negative control paired seras therewith.The serum titer that is tried patient's anti-hSARS antibody sees Table 1.

Table 1

Annotate: * SARS patient

These results show that this coronaviridae newcomer is the keystone pathogen of SARS.

6.6hSARS the sequence of virus

Infect back two days from infecting or do not infect the total RNA of FrHK-4 cell harvesting.With II reversed transcriptive enzyme (Invitrogen) by being recommended in of manufacturer contain the 10pg degenerated primer (5 '-GCCGGAGCTCTGCAGAATTCNNNNNNN-3 ', SEQ ID NO:5; N=A, T, G or C) 20 μ l reaction mixtures in the 100ng purifying RNA is carried out reverse transcription.Pass through then The PCR purification kit is eluted among the 10mM Tris-HCl (pH 8.0) of 30 μ l by manufacturer's operation instruction purifying reverse transcription product.3 μ l purifying cDNA products are joined in the 25 μ l reaction mixtures that contain following composition: 2.5 μ l10xPCR damping fluids, 4 μ l 25mM MgCl ₂, 0.5 μ l10mM dNTP, 0.25 μ lAmpliTaq

Archaeal dna polymerase (Applied Biosystems), 2.5 μ Ci[α- ³²P] CTP (Amersham), 2 μ l10 μ M primers (5 '-GCCGGAGCTCTGCAGAATTC-3 ': SEQ ID NO:6).By following program thermal cycling is carried out in reaction: 94 ℃ 8 minutes, then 94 ℃ 1 minute, 40 ℃ 1 minute, 72 ℃ of circulations of 2 minutes 2 times.After this thermal cycling, carry out 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ circulations of 1 minute 35 times.Get 6 μ lPCR products and carry out 5% denaturing polyacrylamide gel electrophoresis analysis.Gel to the exposure of X-ray sheet, after spending the night, is developed in the exposure of X-ray sheet.The unique PCR product that only identifies in the cells infected sample is separated from gel, with 50 μ l 1x TE buffer solution elution.PCR product with wash-out increases in containing 25 μ l reaction mixtures of following composition more then: 2.5 μ l10xPCR damping fluids, 4 μ l 25mM MgCl ₂, 0.5 μ l10mM dNTP, 0.25 μ l AmpliTaq

Archaeal dna polymerase (Applied Biosystems), 1 μ l10 μ M primer (5 '-GCCGGAGCTCTGCAGAATTC-3 ': SEQ ID NO:6).By following program thermal cycling is carried out in reaction: 94 ℃ 8 minutes, 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ circulations of 1 minute are 35 times then.Use TOPO TA

Test kit (Invitrogen) clone PCR products is transformed into the plasmid that connects in TOP 10 intestinal bacteria (E.coli) competent cells (Invitrogen).PCR inserts fragment to be passed through The cycle sequencing test kit checks order by the suggestion of manufacturer (Applied Biosystems), and the order-checking product is analyzed by automatic sequencer (Applied Biosystems, model 3770).The sequence (SEQ ID NO:1) that obtains is seen Fig. 1.The aminoacid sequence (SEQ ID NO:2) of inferring from the dna sequence dna that obtains shows that it has 57% homology with the polymerase protein of identifying coronavirus.

Similarly, from the aminoacid sequence (being respectively SEQ ID NO:12 and 14) that hSARS virus obtains other two kinds of partial sequences (SEQ ID NO:11 and 13) and infers, see Fig. 8 (SEQ ID NO:11 and 12) and Fig. 9 (SEQ ID NO:13 and 14).

The whole genome sequence of hSARS virus is seen Figure 10 (SEQ ID NO:15).The deduction aminoacid sequence of the SEQ ID NO:15 that obtains with whole three kinds of frames sees that (dna sequence dna is seen SEQ ID NO:16,240 and 737 to Figure 11; Aminoacid sequence is seen SEQ ID NO:17-239,241-736 and 738-1107 respectively).The deduction aminoacid sequence of the complement of the SEQ ID NO:15 that obtains with whole three kinds of frames sees that (dna sequence dna is seen SEQ NO:1108,1590 and 1965 to Figure 12; Aminoacid sequence is seen SEQ ID NO:1109-1589,1591-1964 and 1966-2470 respectively).

6.7 the detection of hSARS virus in the nasopharynx aspirate

At first, by influenza virus A and B, Parainfluenza type 1 virus, 2 types and 3 types, respiratory syncytial virus and adenovirus (the Chan KH in the tachysynthesis fluorescent antigen detection method inspection nasopharynx aspirate (NPA), Maldeis N, Pope W, Yup A, Ozinskas A.Gill J, SetoWH, Shortridge KF, Peiris JSM.Evaluation of Directigen Fly A+B testfor rapid diagnosis of influenza A and B virus intctions.JClin Microbiol.2002; 40:1675-1680), in Mardin Darby Madin-Darby canine kidney(cell line), LLC-Mk2, RDE, Hep-2 and MRC-5 cell, cultivate conventional respiratory pathogen (the Wiedbrauk DL of nasopharynx aspirate, Johnston SLG.Manual of clinical virology.Raven Press, New York.1993).Subsequently, tire RhMK cell (FRhk-4) and A-549 cell are joined in used a series of clones.Directly clinical sample is carried out RT-polymerase chain reaction (RT-PCR) to detect influenza virus A (Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L, Rimmelzwan GF, Osterhaus AD.Detection of influenzaA virus from different species by PCR amplification of conservedsequences in the matrix gene.J Clin Microbiol.2000; 38:4096-101) and human stroma lung virus (HMPV).The primer that is used for HMPV is: the first round, 5 '-AARGTSAATGCATCAGC-3 ' (SEQ ID NO:7) and 5 '-CAKATTYTGCTTATGCTTTC-3 ' (SEQ ID NO:8); And nested primers: 5 '-ACACCTGTTACAATACCAGC-3 ' (SEQ ID NO:9) and 5 '-GACTTGAGTCCCAGCTCCA-3 ' (SEQ ID NO:10).The size of nested PCR product is 201bp.Use at the ELISA of mycoplasma screening cell culture (RocheDiagnostics GmbH, Roche, Indianapolis, USA).

6.7.1.RT-PCR test

To cultivating from two patients' hSARS virus and heredity order-checking back (referring to above 6.6 saving), develop the RT-PCR that is used for from NPA sample detection hSARS virus.Total RNA from clinical sample carries out reverse transcription with random hexamer, cDNA with primer 5 '-TACACACCTCAGCGTTG-3 ' (SEQ ID NO:3) and 5 '-CACGAACGTG-ACGAAT-3 ' (SEQ ID NO:4) is at 2.5mM MgCl ₂Have amplification down (94 ℃ 8 minutes, 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ circulations of 1 minute are 40 times then), described two kinds of primers are based on that the hSARS viral genome makes up.

Typical R T-PCR scheme is summarized as follows:

RNA extracts

By Viral RNA extracts test kit and extract RNA from 140 μ lNPA sample, is eluted in the 50 μ l elution buffers.

Reverse transcription

RNA 11.5μl

0.1M?DTT 2μl

5x damping fluid 4 μ l

10mM?dNTP 1μl

Superscript?II，200U/μl(Invitrogen) 1μl

Random hexamer, 0.3 μ g/ μ l, 0.5 μ l

42 ℃ of reaction conditionss, 50 minutes

94 ℃, 3 minutes

4℃

PCR

The following cDNA that amplification produces by random primer in the 50ul reactant:

cDNA 2μl

10mM?dNTP 0.5μl

10x damping fluid 5 μ l

25mM?MgCl ₂ 5μl

25 μ M forward primers, 0.5 μ l

25 reverse primers, 0.5 μ l

AmpliTaq Polysaccharase, 5U/ μ l (Applied Biosystems) 0.25 μ l

Water 36.25 μ l

Thermal cycle conditions: 95 ℃ 10 minutes, 95 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ circulations of 1 minute are 40 times then.

Primer sequence

Primer is based on RNA RNA-dependent polysaccharase encoding sequence (SEQID NO:1) design of hSARS virus.

Forward primer: 5 ' TACACACCTCAGCGTTG 3 ' (SEQ ID NO:3)

Reverse primer: 5 ' CACGAACGTGACGAAT 3 ' (SEQ ID NO:4)

Product (amplicon) size: 182bp

The real-time quantitative PCR test

By

The miniature test kit of viral RNA (Qiagen) extracts total RNA by manufacturer's explanation from 140 μ l nasopharynx aspirates (NPA).In containing the 20 μ l reaction mixtures of 0.15 μ g random hexamer, 10mmol/L DTT and 0.5mmol/L dNTP, with the RNA sample 200U of 10 μ l wash-outs II reversed transcriptive enzyme (Invitrogen) carries out reverse transcription by explanation.Complementary DNA exists then

Increase in Green I fluorescent reaction (Roche) mixture.Briefly, contain 2 μ l of cDNA, 3.5mmol/L MgCl ₂, 0.25 μ mol/L forward primer (5 '-TACACACCTCAGCGTTG-3 '; SEQ ID NO:3) and 0.25 μ mol/L reverse primer (5 '-CACGAACGTGACGAAT-3 '; SEQ ID NO:4) in the 20 μ l reaction mixtures, with Light-Cycler (Roche) press the PCR program [95 ℃ 10 minutes, then 95 ℃ 10 minutes; 57 ℃ 5 seconds; 72 ℃ of circulations of 9 seconds 50 times] carry out thermal cycling.The plasmid that contains target sequence is as positive control.The fluorescent signal of these reactants extends at each round-robin catches when step finishes.Be the specificity of confirmed test, when off-test, PCR product (184 base pairs) carried out curve analysis (65 ℃ to 95 ℃, 0.1 ℃ of per second).

Clinical effectiveness

Clinical discovery:

All 50 SARS patients are the China race.Other accidental case that they have represented 5 relevant groups of different epidemiology and have met case definition.They on average begin to be in hospital in back 5 days in symptom.Their mean age is 42 years old (23 years old to 74 years old), and the women and the male sex's ratio is 1.3.Wherein 14 people (28%) are medical and nursing work persons, and 5 people (10%) have the visit history to the hospital that seriously breaks out SARS.13 people (26%) are the contact SARS patient of family, the contacted socially SARS patient of other 12 people (24%).4 people (8%) have nearest travelling history to the China's Mainland.

Most of patient's main suit is heating (90%) and breathes hard.Cough and myalgia (table 2) appear in patient more than half.Upper airway symptoms appears in small number of patients, as rhinorrhea (24%) and have a sore throat (20%).Diarrhoea (10%) and appetite stimulator (10%) also have report.Initial audible inspection finds to have only 38% patient vesicular rale and air inlet minimizing to occur.Patient's report of 62% has dry cough.All patients all find the consolidation sign through actinoscopy when seeing and treating patients, comprise 1 zone (36 example), 2 zones (13 example) and 3 zones (1 example).

Table 2

* the trunk maculopapule appears in 1 patient.

Most of patient (98%) does not have leukocytotic sign although have a high fever.Invention Peripheral blood examination is found lymphopenia (68%), oligoleukocythemia (26%), thrombopenia (40%) and anaemia (18%) (table 3).The level of liver parenchyma enzyme alanine aminotransferase (ALT) and muscle enzyme creatinine kinases (CPK) raises in 34% and 26% case respectively.

Table 3

Negative in most of case by cultivation, Detection of antigen and PCR to the conventional microbiological examination that known viruse and bacterium carry out.A blood cultivation that inserts 74 years old male patient of intensive care unit (ICU) is found the intestinal bacteria positive, and this is because the acquired urinary tract infection of hospital.When being admitted to hospital, other two patients from its sputum sample basis, isolate Klebsiella pneumonia (Klebsiellapneumoniae) and Haemophilus influenzae (Hemophilus influenzae).

Give per 24 hours oral 500mg levofloxacins of 9 patients, give other 40 patient's intravenous injections (per 8 hours 1.2g)/oral (375mg, every day three times) amoxicillin with clavulanic acid salt and intravenous injection in per 12 hours/oral 500mg clarithromycin.Give 4 patient's every days twice oral 75mg oseltamivir.Give per 24 hours intravenous injection 2gm ceftriaxones of 1 patient, per 24 hours oral 500mg Azythromycins, every day, twice oral 100mg amantadine covered so that typical and atypical pneumonia are carried out experience.

19 patients develop into and have the desat serious disease of oxygen, need accept grave illness monitoring and ventilation and support.The average fate that begins state of an illness deterioration from symptom is 8.3 days.Symptom begins the back and gave per 8 hours intravenous injection ribavirin 8mg/kg of 49 patients and steroid in average 6.7 days.

Accept with needs that the relevant risk factors of serious concurrent disease that grave illness monitoring and ventilation support are old, lymphopenia, ALT is undermined postpones to give ribavirin and steroid (table 4).All concurrent cases are used ribavirin and steroid therapy after inserting the intensive care unit (ICU), and institute has or not the intercurrent disease example just to use ribavirin and steroid therapy in public ward.As expected, 31 no concurrent cases recoveries from illness or take a turn for the better, and 8 concurrent cases sb.'s illness took a turn for the worse, wherein 1 death when this specification sheets is write.All 50 patients on average accept to monitor 12 days when this specification sheets is write.

Table 4

^*Because case quantity is few, does not carry out multivariate analysis;

2 patients have diabetes, and 1 has the obstructive cardiomyopathy of plumpness, and 1 has chronic active hepatitis B, and 1 has cerebral tumor;

1 patient has essential hypertension;

The § desaturation needs the grave illness monitoring to support;

1 death of ‖.

Separate two viral isolates on one's body from two patients, be accredited as coronaviridae member (vide infra) afterwards.Viral isolates is from a Hong-Kong resident's of 53 years old incision lung biopsy tissue, and another viral isolates is from 42 years old women's in good health in the past nasopharynx aspirate.The Chinese visitor that this 53 years old male sex and one died from SARS from Guangzhou, afterwards has family's contact history of 10 hours.Contact two days later, he heating, uncomfortable, myalgia and headache symptom just occur.There is vesicular rale in lung bottom right district, and the chest radiograph shows corresponding alveolar shade.Hematological examination shows lymphopenia, is 0.7x10 ⁹/ L, total leukocyte and platelet count are normal.ALT (41U/L) and CPK (405U/L) are all impaired.Although he is oral Azythromycin, amantadine and intravenous injection ceftriaxone, both sides lung instillation still occurring increases and carrying out property oxygen desaturation.Therefore, he cut lung biopsy after being admitted to hospital 9 days.Histopathological examination shows that medium matter inflammation, dispersive alveolar cell present that cell gigantism, granular dichromophilism tenuigenin, nucleus increase, kernel is outstanding.There is not cell to demonstrate the typical inclusion body of simplexvirus or adenovirus infection.After undergoing surgery, this patient needs to accept ventilation and grave illness monitoring.Empirical given his intravenous injection ribavirin and hydrocortisone.But he is still dead after being admitted to hospital 20 days.When looking back, in its nasopharynx aspirate, lung biopsy and lung after death, find coronavirus sample RNA.He significantly raises by the antibody titers of anti-self hSARS isolate, rises to 1/1600 from 1/200.

The patient that second place is isolated hSARS virus is 42 years old women in good health in the past.She once arrived the Guangzhou of China's Mainland and went on a journey two days, got back to Hong Kong and occurred heating and symptom of diarrhea after five days.She is carried out physical examination show that there is vesicular rale in lung bottom right district, the chest radiograph shows corresponding alveolar shade.Check and also show oligoleukocythemia (2.7x10 ⁹/ L), lymphopenia (0.6x10 ⁹/ L) and thrombopenia (104x10 ⁹/ L).Although carry out empirical antimicrobial covering with amoxicillin with clavulanic acid salt, clarithromycin and oseltamivir to her, she worsens in the back five days state of an illness of being admitted to hospital, and need accept the monitoring of mechanical ventilation and grave illness and reach five days.She takes a turn for the better gradually subsequently, need not accept ribavirin or steroid therapy.Its nasopharynx aspirate is positive to virus in the RT-PCR test, and she realizes seroconversion, anti-hSARS isolate antibody titers from＜1/50 to＜1/1600.

Virusology is found:

Lung biopsy and nasopharynx aspirate from above-mentioned two patients isolated virus respectively on the FRhk-4 cell.Inoculate back 2 days to 4 days and initial cytopathic effect occurs, but through going down to posterity subsequently, cytopathic effect occurred in 24 hours.Two viral isolates all not with a series of conventional reagent react that is used for the identifying virus isolate, comprise the reagent (DAKO that is used to identify influenza virus A, B, Parainfluenza type 1 virus, 2 types and 3 types, adenovirus and respiratory syncytial virus, Glostrup, Denmark).Described two viral isolates be not used for the RT-PCR test reaction of influenza virus A and HMPV yet, or are being used for the PCR test reaction of mycoplasma.Virus shows that to the ether sensitivity it is an envelope virus.To negative staining (2% phospho-wolframic acid potassium by super centrifugal acquisition, pH 7.0) cell culturing extract carries out submicroscopy and finds, there is many types of envelope virus particle, as if diameter is about 80-90nm (scope of 70-130nm), and its surface morphology can be compared (Fig. 5 A) with the coronaviridae member.Cells infected is carried out the thin section submicroscopy show that it is the virion (Fig. 5 A and 5B) of 55-90nm that diameter is arranged in the slide wall vesicle in the tenuigenin.At the also visible virion of cell surface.Overall discovery is consistent with the cell infection that the coronavirus coe virus causes.

The described 53 years old male sex's lung biopsy thin section electron photomicrograph shows, contains the 60-90nm virion in the tenuigenin of its furfur cell.These virions the size with morphology on to observed virion similar (Fig. 4) in from two patients' cell culture and virus isolate.

The RT-PCR product that produces in the random primer RT-PCR test is analyzed, the peculiar band of finding in the virus infection sample is cloned and checked order.In 30 clones that check, identify the clone of 646 base pairs (SEQ ID NO:1) that contain unknown source.This dna fragmentation is carried out sequencing analysis show, the virus of this sequence and coronaviridae family has weak homology (data do not provide).(215 amino acid: SEQ ID NO:2) but the RNA polymerase with bovine coronavirus and murine hepatitis virus has homology (57%) highly to the aminoacid sequence of unknown nucleotide sequence deduction from then on, proves conclusively this virus and belongs to coronaviridae family.The phylogenetics analysis of protein sequence shows, though this virus is closely related but distinct virus (Fig. 5 A and 5B) most with coronavirus II class.

646 base-pair sequences according to this isolate have designed the Auele Specific Primer that is used to detect new virus, detect in clinical sample this hSARS viral genome being carried out RT-PCR.From 44 nasopharynx aspirate samples that 50 SARS patients obtain, 22 samples have hSARS RNA sign.In 18 inspected faecal samples, there are 10 can detect viral RNA.The specificity of RT-PCR reaction is confirmed by the positive RT-PCR amplified production of selecting is checked order.In the RT-PCR test, has reactivity from none in no related disorders patient's 40 nasopharynx aspirates and the fecal sample.

For determining the dynamicrange of real-time quantitative PCR, preparation contains the serial dilution thing of the plasmid DNA of target sequence, and it is carried out the real-time quantitative PCR test.Shown in Fig. 7 A, this test can detect few target sequence to 10 copies.By comparison, in the water contrast, do not observe signal (Fig. 7 A).In the SARS patient that 29 serology are confirmed, there are 23 to observe positive signal.In all these positive cases, observe and the corresponding unique PCR product (T of the signal of positive control _m=82 ℃) (Fig. 7 B, data do not provide).These data show that this test has the specificity of height to target.In these reactions the copy number of target sequence from 4539 to being less than 10.Therefore, in 1ml NPA sample, can find up to 6.48x10 ⁵This virus sequence of individual copy.There are 5 can before seroconversion, collect the NPA sample in the above-mentioned positive case.3 in these samples detect viral RNA, show this test even just can detect virus at the early stage of infection outbreak.

Be further to confirm the specificity of this test, patient's the NPA sample of collecting healthy people (n=11) and infection adenovirus (n=11), respiratory syncytial virus (n=11), human stroma lung virus (n=11), influenza virus A (n=13) or influenza virus B (n=1) is as negative control.All these samples are except that one, and test-results is all negative.The false positive case is negative in test subsequently.Comprise false-positive case, the sensitivity of real-time quantitative PCR test is 79%, and specificity is 98%.

Epidemic data shows that droplet transmission is one of main route of transmission of this virus.This research detects the virus sequence of live virus and high copy from the NPA sample, C﹠S's spittle of clearly supporting SARS patient may be the main source of this Vector of infection.What is interesting is have in 4 faecal samples from SARS patient in this research 2 be positive in test (data do not provide).Detecting virus in the ight soil shows and may have other route of transmission.Relevant being pointed out that, some ani mal coronavirus is propagated (McIntoshK., 1974, Coronaviruses:a comparative review.Current Top MicrobiolImmunol.63:85-112) by fecal oral route.But, require further study to check the virus in the ight soil whether to be infectious.

Except that this hSARS virus, also have two kinds of known human corona virus's serogroupss (229E and OC43) (Hruskova J. etc. at present, 1990, Antibodies to human coronaviruses229E and OC43in the population of C.R., Acta Virol.34:346-52).The primer pair that is used for this test does not have homology with the 229E strain.Owing in Genebank, can not obtain corresponding OC43 sequence, not know the whether therewith strain generation cross reaction of these primers.But the sequential analysis that obtainable sequence in other zone of OC43 pol gene is carried out shows that the Novel Human virus relevant with SARS is completely different with OC43 in heredity.In addition, the primer that uses in this research and any sequence of known coronavirus all do not have homology.Therefore, unlikely meeting of these primers and OC43 strain generation cross reaction.

Report, except that described novel pathogenic agent, also identify stroma lung virus (Center for Disease Control and Prevention, 2003, Morbidity and Mortality Weekly Report 52:269-272) on one's body some SARS patient.In any patient of this research, do not detect any sign (data do not provide) that stroma lung virus infects, show that the novel hSARS virus of the present invention is the Primary Actor in the SARS pathogeny.

IFA:

35 parts of signs (referring to Fig. 3) with anti-hSARS antibody are arranged in 50 parts of up-to-date serum samples from SARS patient.Seroconversion takes place in 27 patients that can obtain paired acute phase and convalescence serum or its antiviral antibody titre all improves＞and 4 times.Also detect with other 5 pairs of serum of other SARS patient of outgroup from this study group, socially SARS patient is taken a sample widely, seroconversion takes place in all.80 parts of serum and 200 normal blood donors from respiratory tract disease patient or other disease patient all do not have detectable antibody.

If detect the evidence that viral RNA all is considered to infect hSARS virus to the HP-CV seropositivity or in NPA or ight soil in the single serum, have 45 to have the infection sign among 50 patients so.In the middle of 5 patients, have only a patient to accept serum test after＞14 days in the clinical symptom outbreak without any the virusology evidence of coronaviridae virus infection.

6.8 be used to detect the quantitative of hSARS virus

Test

6.8.1. material and method

Patient and sample collection

Use from 50 storage clinical samples that satisfy the patient of the clinical WHO case definition of SARS (http://www.who.int/csr/sars/casedefinition/en/) in this research, these patients' diagnosis confirms by seroconversion subsequently.Begin from symptom went in 1-3 days above-mentioned collection NPA sample (Poon etc., 2003, Clin.Chem.49:953-955).Use from no related disorders patient's NPA sample in contrast.

RNA extracts and reverse transcription

With The miniature test kit of viral RNA (Qiagen) extracts RNA by manufacturer's explanation from clinical sample.In above-mentioned conventional RT-PCR test, 140 μ l NPA are used for RNA and extract.In improved RNA extraction scheme, 540 μ l NPA are used for RNA and extract.The RNA that extracts finally is eluted to 30 μ L not to be had in the RNA enzyme water, is stored in-20 ℃.Use the total RNA of random hexamer reverse transcription then from clinical sample.

The conventional PCR that is used for SARS-CoV

Conventional PCR test is carried out described in the 6.7.1. joint.

Be used for the real-time quantitative PCR test of SARS-CoV

In this research, use the real-time quantitative PCR of specificity at SARS-CoV 1b district.By

The PCR Core Reagent test kit complementary DNA that in 7000 Sequence DetectionSystem (Applied Biosystems), increases.Briefly, amplification 4 μ l cDNA:0.625U AmpliTaq in containing 25 μ l reactants of following compositions

Polysaccharase (Applied Biosystems), 2.5 μ l 10x Buffer A, 0.2mMdNTP, 5.5mM MgCl ₂, 2.5U UNG and 1x primer-probe mixture (Assays by Design, Applied Biosystems).Primer sequence is 5 '-CAGAACGCTGTAGCTTCAAAAATCT-3 ' (SEQ ID NO:2471) and 5 '-TCAGAACCCTGTGATGAATCAACAG-3 ' (SEQ ID NO:2472), probe is 5 '-(FAM) TCTGCGTAGGCAATCC (NFQ)-3 ' (SEQ ID NO:2473; FAM, the 6-Fluoresceincarboxylic acid; NFQ, no fluorescence quencher).Reaction was hatched 2 minutes at 50 ℃ at first, hatched 10 minutes at 95 ℃ then.Reactant carries out thermal cycling 45 times (95 ℃ 15 seconds, 60 ℃ 1 minute) then.The plasmid that contains target sequence is as positive control.

6.8.2. result

Totally 50 separation NPA samples that its disease that the SARS patient that research confirms from serology collects is initial 3 days.Among them, 11 (22%) is positive (referring to the 6.7.1 joint) (table 5) in the conventional RT-PCR test that we report previously.

Table 5

^*Total verification and measurement ratio of test and conventional RT-PCR test have significant difference (McNemar check, P＜0.001)

⁺Total verification and measurement ratio of test has significant difference (McNemar check, P＜0.0001) with the conventional RT-PCR test of carrying out with improvement RNA extraction scheme

Our inference, SARS-CoV RT-PCR detects and can be increased to 560 μ l from 140 μ l by the extraction volume with initial NPA sample at the early stage Wheat Protein of disease and be enhanced.Use this improved RNA extraction scheme, the susceptibility of traditional RT-PCR test is doubled to 22/50 (table 5) from 11/50.Total verification and measurement ratio and our first-generation RT-PCR scheme of improving the RT-PCR scheme have significant difference (McNemar check, P＜0.001, table 5).For 30 negative control samples, observe a false positive results.Adopt RNA to extract and improve, conventional RT-PCR is respectively 44.0% and 96.6% to the susceptibility and the specificity of the sample that the disease first three days is collected.

For further improving the detection of SARS-CoV in the early stage sample of morbidity, we have adopted extremely sensitive real-time quantitative test carrying out SARS-CoV to detect (Figure 14).Adopt improved RNA extraction scheme, in real time 40 positive (Figure 15 and table 5) of 50 NPA samples in the test.Total verification and measurement ratio and other two kinds of tests of improving the RT-PCR scheme have significant difference (McNemar check, P＜0.0001, table 5).Specifically, it is 63% positive in the real-time quantitative RT-PCR test that disease begins the 1st day isolating NPA sample.By comparison, the 1st day isolating sample do not have positive in traditional RT-PCR test.For 2-3 days isolating samples, surpass 81% sample positive (table 5) in quantitative test.Adopt improved RNA extraction scheme and real time pcr, quantitative test is respectively 80% and 100% to the susceptibility and the specificity of early stage SARS sample.

Test in real time also allows the quantitatively virus load (1 copy/reaction=27.8 copy/ml NPA samples) of these clinical samples.As shown in Figure 16, the progress of disease causes virus load rising (blank bar) among the NPA.In addition, we have further investigated the virus load (Figure 16, grey bar) of the negative clinical sample (N=39) in our first-generation RT-PCR test.As expectation, the virus load of these clinical samples (grey bar) is more much lower than whole comprehensive virus load (blank bar).

6.8.3. discuss

This target of studying us is to set up the hypersensitivity RT-PCR test that is used to detect SARS-CoV.Specifically, we concentrate on and detect symptom and begin the 1-3 days SARS-CoV RNA in the isolating sample.Use the conventional RT-PCR test of our first-generation, only have these samples of 22% to show and have SARS-CoV RNA.In order to set up more responsive test, we have improved the RNA extracting method, and have adopted quantitative technique in our current research.Be increased to 540 μ l by the initial volume that will be used for the RNA extraction from 140 μ l, the ratio of positive case is increased to 44%.In addition, by further use the real-time quantitative PCR technology in improving test, 80% early stage SARS sample becomes positive.The more important thing is that the use of 5 nuclease probes can minimize false positive rate in the real-time quantitative test, this is because signal specificity increases.Combine, the result of this research shows that we allow to diagnose in early days and exactly SARS at improved RT-PCR test.

The quantitative result that we improve RT-PCR test further provides the information about SARS-CoV virus load in these clinical samples.Our result shows that virus load increases with the progress of disease.For negative sample in first-generation RT-PCR test, all contain the viral RNA (Figure 15 and 16) of minute quantity.This behavior explains why these samples of great majority are negative when the first-generation RT-PCR test of using us.What is interesting is that for positive sample in first-generation test, some contains the viral RNA (Figure 16) of very high amount.

In a word, be used for original sample volume and the use real-time quantitative PCR technology that RNA extracts by increase, we have set up responsive and RT-PCR test accurately is used for identifying immediately SARS-CoV.What expect is to adopt this fast diagnosis method, the control of the instant evaluation meeting promotion disease of this pathogenic agent and the system of immediate treatment.

6.9. clinical observation and discussion

Breaking out of SARS is uncommon in many aspects, especially occurs the pneumonia patient medical and nursing work person with concentrating during family contacts.In the middle of these a series of SARS patients, the inspection of the conventional pathogenic agent of atypical pneumonia is turned out to be feminine gender.But from respectively available from isolating the virus that belongs to coronaviridae family two SARS patients' lung biopsy and the nasopharynx aspirate.This virus is not closely related with any known human or animal coronavirus or Orbivirus on phylogenetics.This analysis shows that based on the 646 base pair fragments (SEQID NO:1) of pol gene this virus is relevant with bovine coronavirus with antigen 2 classes and the murine hepatitis virus of coronavirus.But, the coronavirus coe virus can carry out the allos reorganization in virus family inside, so be necessary the genomic other parts of new virus are carried out genetic analysis, define essence (the Holmes KV.Coronaviruses.EdsKnipe DM of this virus then more for certain, Howley PM Fields Virology, the 4th edition, Lippincott Williams﹠amp; Wilkins, Philadelphia, 1187-1203 page or leaf).Biology, genetics and clinical data are combined, show that this new virus is not any of two kinds of known person coronavirus.

The most of patient (90%) who suffers from the SARS of clinical definition has this viral serology of infection and RT-PCR evidence.Contrast does not therewith have antibody or viral RNA to detect in healthy people's contrast.All 27 patients that can obtain acute phase and convalescence serum show that all the antibody titers of anti-hSARS virus raises, and this has strengthened following argument, and promptly infecting this virus recently is the developing necessary factor of SARS.In addition, also show seroconversion after testing from all 5 pairs of acute phases of the patient of other hospital of Hong Kong and convalescence serum to virus.Do not show that the serology of hSARS virus infection or 5 patients of virusology evidence need later on convalescence serum is detected, whether seroconversion also takes place to determine them.But if the definition of clinical case is never clear and definite, hSARS virus shows still very significantly with the consistence of SARS clinical definition.

No matter be by RT-PCR or according to the rising of anti-HMPV antibody titers, none detects the sign that HMPV infects among these patients.In our SARS patient's group, do not detect other pathogenic agent all the time.Therefore, this hSARS virus is to cause the reason of SARS or the prerequisite of this disease progression probably.The problem that whether has other microbiological factor or other cofactor to have an effect in this advancing of disease is still waiting investigation.

Coronaviridae family comprises coronavirus genus and Orbivirus.They are enveloped RNA viruses, can cause the human and animal ill.Human corona virus 229E that knew in the past and OC43 type are major cause (Holmes KV.Coronaviruses.Eds Knipe DM, Howley PM Fields Virology, the 4th edition, the LippincottWilliams﹠amp that causes common cold; Wilkins, Philadelphia, 1187-1203).But, though coronavirus can cause pneumonia (El-Sahly HM sometimes in old man, ewborn infant or immunocompromised patient, Atmar RL, Glezen WP, Greenberg SB.Spectrum of clinical illness inhospitalizied patients with " common cold " virus infections.Clin Infect Dis.2000; 31:96-100; With Foltz EJ, Elkordy MA.Coronavirus pneumoniafollowing autologous bone marrow transplantation for breast cancer.Chest1999; 115:901-905), have and report that they are major reasons of pneumonia among the new recruit of army, in some research, account for case (Wenzel RP up to 30%, Hendley JO, Davies JA, Gwaltney JM, Coronavirus infections in military recruits:Three-yearstudy with coronavirus strains OC43and 229E.Am Rev Respir Dis.1974; 109:621-624).The human corona virus can infect neurone, in the brain of multiple sclerosis patients, detected viral RNA (Talbot PJ, Cote G is during Arbour N.Humancoronavirus OC43 and 229E persistence in neural cell cultures and humanbrains.Adv Exp Med Biol. delivers).On the other hand, some ani mal coronavirus (for example transmissible gastroenteritis of swine virus, murine hepatitis virus, avian infectious bronchitis virus) can cause their hosts separately that respiratory tract disease, gastrointestinal illness, sacred disease or hepatopathy (McIntosh K.Coronaviruses:a comparative review.Current TopMicrobiol Immunol.1974 takes place; 63:85-112).

We are described clinical manifestation and the complication of SARS for the first time.Coronavirus pneumonia patient less than 25% has upper airway symptoms.As what atypical pneumonia was expected, respiratory symptom and positive auscultation result are very unbecoming with chest radiograph result.Gastrointestinal symptom appears in 10% patient.Relevant is, coronavirus RNA can detect in some patient's faecal samples, and the known diarrhoea of coronavirus relevant (CaulEO, Egglestone SI.Further studies on human enteric coronaviruses ArchVirol.1977 with animal and human's class; 54:107-17).Liver function disorder, oligoleukocythemia, remarkable lymphopenia, thrombopenia reach the rate occurred frequently that develops into adult respiratory distress syndrome subsequently and show that this hSARS virus has caused serious general inflammatory damage.Therefore carrying out immunomodulatory by steroid is very important with the antiviral therapy of assisting ribavirin.In this, it is appropriate (Cheung CY that the same supposition serious human diseases relevant with H 5 N 1 avian influenza hypotype (giving human another kind of virus from the animal cross infection recently) has the immunopathology composition, Poon LLM, Lau ASY etc., Induction of proinflammatory cytokines inhuman macrophages by influenza A (H5N1) viruses:a mechanism for theunusual severity of human disease.Lancet 2002; 360:1831-1837).The same with the H5N1 disease, serious SARS patient also is the grownup, its lymphopenia is more remarkable, and has respiratory tract feature (table 4) the Yuen KY of organ dysfunction in addition, Chan PKS, Peiris JSM etc., Clinical features and rapid viral diagnosis of human diseaseassociated with avian influenza A H5N1 virus.Lancet 1998; 351:467-471).What deserves to be explained is, begin the window of opportunity of having an appointment 8 days from disease to respiratory insufficiency.The severe complications case is used strong relevant with the delay of potential disease and ribavirin and Steroid treatment.According to the clinical experience that we obtain from initial case, do not have basically when being admitted to hospital that we implement the aforesaid combination therapy very early in the case afterwards of complication.Adopt this treatment plan, general mortality rate has only 2% when this specification sheets is write.In 19 complication cases, also have 8 significantly reaction not occur.Because dosage and initial treatment time are inconsistent, impossible detailed analysis is to the therapeutic response of this assembled scheme.

The relevant other factors of serious disease is to contact and ill by family therewith, and this is attributable to high dosage or continues to be exposed to virus and have potential disease.

In this clinical description of doing is serious case about being hospitalized for treatment basically.We are also without any the complete clinical range data that infects about the coronaviridae that occurs in society and the outpatient service at present.The validity of diagnostic test described herein will help to overcome the above problems.In addition, this also allows to solve period about virus shedding in the rehabilitation (and infectivity), the virus existence and the problems such as generation of virus shedding in latent period in other body fluid and movement.

Present epidemic data as if show virus by the spittle or directly and indirect contact propagate, pass through airborne transmission though can not get rid of in some cases.The discovery infective virus has been supported this argument in respiratory tract.Preliminary evidence hints that also virus may come off in ight soil.But, what deserves to be explained is that detecting of viral RNA can not prove that virus has viability or infectivity.If detect live virus in ight soil, this may be another potential route of transmission that need take in.Can appropriately be pointed out that, some ani mal coronavirus by fecal oral route propagate (McIntosh K., Coronaviruses:a comparativereview.Current Top Microbiol Immunol.1974,63:85-112).

In a word, this report provides following evidence, and promptly the virus of coronaviridae family is the pathogenic factor of SARS.The quantitative Diagnosis test of the invention discloses fast, sensitivity and specificity being identified hSARS virus.

7. preservation

The sample that separates hSARS virus is deposited in the Chinese typical culture center (CCTCC) that is positioned at Wuhan University (Chinese Wuhan 430072) according to the microbial preservation budapest treaty on April 2nd, 2003, the preservation searching number that gives is CCTCC-V200303, its by reference integral body be attached to herein.

8. market potential

Existing energy large scale culturing hSARS virus, this makes can develop aforesaid various diagnostic test and develop vaccine and the antiviral that can effectively prevent, improve or treat SARS.In view of this severity of disease and at global rapid spread, the demand of the diagnostic test, therapy and the vaccine that are used to resist this disease is obviously risen in worldwide probably.In addition, this virus contains clinical and the of crucial importance and valuable genetic information of research application.

9. equivalence

Those of ordinary skills only adopt normal experiment, just will recognize the many equivalence that maybe can determine specific embodiments described herein.This equivalence is forgiven by following claims.

All publications, patent and the patent application of mentioning in this manual integral body by reference is attached in this paper specification sheets, and its degree clearly and is individually pointed out that integral body is attached to herein by reference as each independent publication, patent or patent application.

This paper should not be interpreted as admitting Here it is prior art of the present invention to quoting or discussing of reference.

Claims

1. isolated nucleic acid molecule, the sequence of described nucleic acid molecule is SEQ ID NO:2471,2472 nucleotide sequences or its complementary sequence.

2. isolated nucleic acid molecule, the sequence of described nucleic acid molecule is SEQ ID NO:2474,2475 nucleotide sequences or its complementary sequence.

3. isolated nucleic acid molecule, the sequence of described nucleic acid molecule is SEQ ID NO:2473,2476 nucleotide sequences or its complementary sequence.

4. a cover nucleic acid molecule is used for biological sample in preparation and detects purposes in the medicine that hSARS virus exists, and a described cover nucleic acid molecule is that nucleotide sequence is SEQ ID NO:2471,2472 or 2473 nucleic acid molecule, when detecting:

(a) use the nucleic acid of nucleotide sequence as the primer amplification hSARS virus of SEQ ID NO:2471 and/or 2472;

(b) use the probe in detecting nucleic acid of nucleotide sequence as SEQ ID NO:2473.

5. a cover nucleic acid molecule is used for biological sample in preparation and detects purposes in the medicine that hSARS virus exists, and a described cover nucleic acid molecule is that nucleotide sequence is SEQ ID NO:2474,2475 or 2476 nucleic acid molecule, when detecting:

(a) use the nucleic acid of nucleotide sequence as the primer amplification hSARS virus of SEQ ID NO:2474 and/or 2475;

(b) use the probe in detecting nucleic acid of nucleotide sequence as SEQ ID NO:2476.

One cover derive from the hSARS nucleotide sequence primer in the purposes that is used for preparing the medicine of identifying hSARS virus infection object, described primer is each a nucleic acid molecule among the claim 1-3, when detecting:

(a) obtain total RNA from biological sample from object;

(b) the total RNA of reverse transcription is to obtain cDNA;

(c) with described primer cDNA is carried out the PCR test.

7. a cover nucleotide sequence is that the primer of SEQ ID NO:2471 and/or 2472 is used for identifying the purposes of the medicine of hSARS virus infection object in preparation, when detecting:

(a) obtain total RNA from biological sample from object;

(b) the total RNA of reverse transcription is to obtain cDNA;

(c) with described primer cDNA is carried out the PCR test.

8. the purposes of claim 7 further comprises the probe of the product that is used to detect the PCR test in the described medicine.

9. the purposes of claim 8, wherein said probe is the nucleic acid molecule that nucleotides sequence is classified SEQ IDNO:2473 as.

10. a cover nucleotide sequence is that the primer of SEQ ID NO:2474 and/or 2475 is used for identifying the purposes of the medicine of hSARS virus infection object in preparation, when detecting:

(a) obtain total RNA from biological sample from object;

(b) the total RNA of reverse transcription is to obtain cDNA;

(c) with described primer cDNA is carried out the PCR test.

11. the purposes of claim 10 further comprises the probe of the product that detects the PCR test in the described medicine.

12. the purposes of claim 11, wherein said probe are the nucleic acid molecule that nucleotides sequence is classified SEQ IDNO:2476 as.

13. a test kit, described test kit comprise one or more isolated nucleic acid molecule in one or more containers, described nucleic acid molecule is made up of the nucleotide sequence that is selected from SEQ ID NO:2471, SEQ IDNO:2472 and SEQ ID NO:2473.

14. a test kit, described test kit comprise one or more isolated nucleic acid molecule in one or more containers, described nucleic acid molecule is made up of the nucleotide sequence that is selected from SEQ ID NO:2474, SEQ IDNO:2475 and SEQ ID NO:2476.