CN1768140A - A high-troughput diagnostic assay for the human virus causing severe acute respiratory syndrome (SARS) - Google Patents

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

High-throughput 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 60/465 that on April 25th, 2003 submitted to, the U.S. Provisional Application 60/470 that on May 14th, 738 and 2003 submitted to, 935, 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 1.6MB file (V9661077.APP).

1. introduce

The present invention relates to high-throughput diagnostic test to the virus (" hSARS virus ") that causes human severe acute respiratory syndrome (SARS).Specifically, the present invention relates to high-throughput reverse transcription-pcr diagnostic and measure SARS associated coronavirus (SARS-CoV).Test method of the present invention is rapid and reliable detection method, can be used for diagnosing and monitoring the propagation of SARS.The inventive method has been eliminated false negative result, and provides highly sensitive for test.The invention further relates to the nucleotide sequence and the part thereof that are used to diagnose SARS.The invention still further relates to the nucleotide sequence and the part thereof that are used to estimate the SARS genetic polymorphism.Nucleotide sequence of the present invention comprises hSARS virus (nucleocapsid) N-gene and S-gene order.The present invention relates to be used to detect the diagnostic kit that comprises nucleic acid molecule of hSARS virus N-gene or S-gene.The invention still further relates to the hSARS virus N-gene of deduction and the aminoacid sequence of S-gene product.The invention still further relates to N-gene and the purposes of S-gene product in diagnostic method.The present invention also comprises the anti-N-gene that comprises generation or the diagnostic test method and the test kit of S-gene product antibody.

2. background of invention

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).The patient who suffers from SARS shows various clinical symptom, comprises heating (38 degrees centigrade or above surpass 24 hours), discomfort, shiver with cold, headache and body bitterly.Chest X-ray show can be compatible with pneumonia change.Other symptom comprises cough, short of breath or expiratory dyspnea.To on May 3rd, 2003, in Hong Kong totally 1621 cases take place altogether, 179 people's death account for 26% and 41% of whole world report case (6234) and dead (435) respectively.Because this disease has hyperinfection, and propagates in daily routines, be starved of exploitation a kind of fast and the reliable diagnostic detection method to monitor and to control this disease.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 worker colony suffers from this disease.In addition, sick therewith the infected closely the philtrum of contact a plurality of pneumonia case colony appears.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.The present inventor's (being called for short the inventor) 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.In addition, the diagnosis detecting method of other gene such as 1b-gene can not be used for accurately diagnosing SARS in the detection hSARS virus.This disease is by the initial name (" SARS ") of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome).Therefore this virus mutation and change very soon diagnoses very difficulty of SARS, up to the peculiar zone of from SARS patient, isolating this virus as disclosed herein by the inventor, and the N-gene of hSARS virus and S-gene.Promptly the invention discloses the Special Areas that uses in this viral genome fast, accurately, reliable and specificity identifies the diagnostic test method of hSARS virus.The present invention can be used for clinical and the scientific research purposes.In addition, the invention provides the high throughput test method, can be used as the sensitive method that diagnosis and monitoring SARS propagate.

3. summary of the invention

The present invention is based on the evaluation of the inventor to the peculiar zone of hSARS virus, specifically, is the virus genomic 3 ' zone of hSARS, is particularly useful for diagnostic test to detect hSARS virus (nucleocapsid) the N-gene of hSARS.Specifically, the N-gene is used for diagnosing SARS to be because compare with other gene of hSARS virus, and the N-gene has maximum copy numbers when virus infection, especially when lysis.Therefore, the nucleotide sequence of hSARS virus N-gene especially can be used for the quick and reliable diagnostic test to hSARS virus.In addition, the inventive method has been eliminated false negative result, and has improved the sensitivity of test.

Described virus is from separating in breaking out the patient who suffers from SARS in the serious atypical pneumonia of China recently.This isolated viral is that straight polarity has the coating single strand RNA virus, belongs to nest virus (Nidovirales) order coronaviridae (Coronaviridae).HSARS virus is very huge RNA viruses, is made up of about 29,742 Nucleotide.The complete genome group sequence of hSARS virus is deposited in Genbank, and NCBI, its searching number are AY278491 (SEQID NO:15), and it is attached to herein by reference.Isolating hSARS virus is deposited in Chinese typical culture collection 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.Equally, the complete genome group sequence of hSARS virus CCTCC-V200303 and feature thereof be disclosed in this paper simultaneously acting on behalf of in the U.S. Patent application that file number is V9661.0069 of submitting on March 24th, 2004, its by reference integral body be attached to herein.This virus mutation and change very soon, thereby make very difficulty of diagnosis SARS.The inventor has designed the diagnostic test method that detects N-gene nucleotide series or albumen existence, identifies hSARS virus with quick, accurate and specificity.In addition, the inventor has designed the test method of diagnostic assays S-gene nucleotide series or albumen existence, to measure the genetic polymorphism of hSARS virus.Therefore, the present invention relates to detect the method for hSARS virus N-gene and S-gene nucleotide series.

The invention provides the test method of rapid and reliable detection hSARS virus.In preferred embodiments, the detection of hSARS virus is included in polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), DNA analysis, RNA analysis or other nucleic acid hybridization and uses nucleic acid molecule of the present invention to detect hSARS nucleic acid.In one embodiment, the invention provides and detect in biomaterial such as cell, nasopharynx aspirate, phlegm, blood, saliva, urine, excrement etc. that hSARS virus of the present invention exists, the active or method expressed.In preferred embodiments, biomaterial is nasopharynx aspirate or excrement.The rising of hSARS virus activity or expression relative comparison sample or reduction can be measured by the detection reagent that makes the biomaterial contact can directly or indirectly detect the existence of hSARS virus, activity or expression in the sample.In a specific embodiments, detection reagent is a nucleic acid molecule of the present invention.

The invention still further relates to the method for identifying hSARS virus infection object, described method comprises from the biological sample of this 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 nucleotide sequence with a cover measures.In preferred embodiments, primer derives from (nucleocapsid) N-gene.In most preferred embodiment, primer comprises SEQ ID NO:2475 and/or 2476 or SEQ ID NO:2480 and/or 2481 nucleotide sequences.In a further preferred embodiment, primer derives from (furcella) S-gene.In a more preferred embodiment, primer comprises SEQ ID NO:2477 and/or 2478 nucleotide sequences.

The invention still further relates to the purposes of sequence information in diagnosis and methods of treatment of isolating virus.In a specific embodiments, the invention provides the nucleic acid molecule that is suitable as primer, described nucleic acid molecule comprise or by SEQ ID NO:1,11,13,15,2471 or 2473 nucleotide sequences or its complementary sequence or at least the part of its nucleotide sequence form.In most preferred embodiment, primer comprise SEQ ID NO:2475 and/or 2476 or SEQ IDNO:2480 and/or 2481 nucleotide sequences to detect the N-gene.In another most preferred embodiment, primer comprises SEQ ID NO:2477 and/or 2478 nucleotide sequences to detect the S-gene.In another embodiment, the invention provides the nucleic acid molecule that is fit to the hSARS nucleic acid hybridization, include but not limited to that the probe of PCR primer, reversed transcriptive enzyme primer, DNA analysis or other nucleic acid hybridization analysis to detect hSARS nucleic acid, for example comprise or are made up of SEQ ID NO:1,11,13,15,2471,2473,2475,2476,2477,2478,2480 or 2481 nucleotide sequences or its complementary sequence or its part.In preferred embodiments, amplification comprises the segmental primer of (the 18057-18222 position Nucleotide of SEQ ID NO:15 or its part) 1b gene, (the 21920-22107 position Nucleotide of SEQ ID NO:15 or its part) M-gene and (the 28658-28883 position Nucleotide of SEQ ID NO:15 or its part) N-gene, can be used for the probe of the synthetic hSARS of detection nucleic acid.In a specific embodiments, the invention provides the diagnostic kit that comprises nucleic acid molecule, it is suitable for detecting the N-gene of hSARS.In a specific embodiments, the N-gene comprises SEQ ID NO:2471 nucleotide sequence.In a specific embodiments, nucleic acid molecule comprises SEQ ID NO:2475 and/or 2476 or SEQ ID NO:2480 and/or 2481 nucleotide sequences.In preferred embodiments, diagnostic kit also comprises the contrast of the 1b gene that is used to increase.In a specific embodiments, the primer of the 1b gene that is used to increase is SEQ ID NO:3 and/or 4.In another embodiment, diagnostic kit also comprises the internal contrast that uses pig beta-actin gene.In a specific embodiments, the primer of the beta-actin gene that is used to increase is SEQ ID NO:2482 and/or 2483.

In a specific embodiments, the invention provides the diagnostic kit that comprises nucleic acid molecule, this nucleic acid molecule is suitable for detecting the S-gene of hSARS.In a specific embodiments, the S-gene comprises SEQ ID NO:2473 nucleotide sequence.In a specific embodiments, nucleic acid molecule comprises SEQ ID NO:2477 and/or 2478 nucleotide sequences.The present invention also comprises in whole or in part by described nucleotide sequence coded chimeric or recombinant virus.

In another embodiment, the invention provides the nucleic acid molecule that comprises SEQ ID NO:1,11,13,2471 and/or 2473 nucleotide sequences.In a specific embodiments, the invention provides isolated nucleic acid molecule, this nucleic acid molecule comprises or is made up of SEQ ID NO:1 nucleotide sequence or its complementary sequence or its part, at least 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600 or more continuous nucleotides of preferred SEQ ID NO:1 nucleotide sequence or its complementary sequence.In another embodiment, the invention provides isolated nucleic acid molecule, this nucleic acid molecule comprises or is made up of SEQ ID NO:11 nucleotide sequence or its complementary sequence or its part, at least 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,700,750,800,850,900,950,1000,1050,1100,1150,1200 or more continuous nucleotides of preferred SEQ ID NO:11 nucleotide sequence or its complementary sequence.In another specific embodiments, the invention provides isolated nucleic acid molecule, this nucleic acid molecule comprises or is made up of SEQ ID NO:13 nucleotide sequence or its complementary sequence or its part, at least 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700 or more continuous nucleotides of preferred SEQ ID NO:13 nucleotide sequence or its complementary sequence.In another embodiment, the invention provides isolated nucleic acid molecule, this nucleic acid molecule comprises or is made up of SEQ ID NO:15 nucleotide sequence or its complementary sequence or its part, at least 5 of preferred SEQ ID NO:15 nucleotide sequence or its complementary sequence, 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 more continuous nucleotides.In another embodiment, the invention provides isolated nucleic acid molecule, this nucleic acid molecule comprises or is made up of SEQ ID NO:2471 nucleotide sequence or its complementary sequence or its part, at least 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 more continuous nucleotides of preferred SEQ ID NO:2471 nucleotide sequence or its complementary sequence.In another embodiment, the invention provides isolated nucleic acid molecule, this nucleic acid molecule comprises or is made up of SEQ ID NO:2473 nucleotide sequence or its complementary sequence or its part, at least 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 or more continuous nucleotides of preferred SEQ ID NO:2473 nucleotide sequence or its complementary sequence.In addition, in another embodiment, the invention provides isolated nucleic acid molecule, this nucleic acid molecule under the stringent condition of this paper definition with the making nucleic acid molecular hybridization with SEQ ID NO:1,11,13,15,2471 or 2473 sequences or its complementary sequence.In one embodiment, the invention provides isolated nucleic acid molecule with the coding strand antisense of nucleic acid of the present invention.In another embodiment, the invention provides isolated polypeptide or albumen by nucleic acid molecule encoding, this nucleic acid molecule comprises or is made up of the nucleotide sequence of at least 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600 or more continuous nucleotides of SEQ IDNO:1 nucleotide sequence or its complementary sequence.In another specific embodiments, the invention provides isolated polypeptide or albumen by nucleic acid molecule encoding, this nucleic acid molecule comprises or is made up of the nucleotide sequence of at least 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 more continuous nucleotides of SEQ ID NO:11 nucleotide sequence or its complementary sequence.In another specific embodiments, the invention provides isolated polypeptide or albumen by nucleic acid molecule encoding, this nucleic acid molecule comprises or is made up of the nucleotide sequence of at least 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700 or more continuous nucleotides of SEQ ID NO:13 nucleotide sequence or its complementary sequence.In another specific embodiments, the invention provides isolated polypeptide or albumen by nucleic acid molecule encoding, this nucleic acid molecule comprises or by 5 of SEQ ID NO:15 nucleotide sequence or its complementary sequence at least, 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 form.In another specific embodiments, the invention provides isolated polypeptide or albumen by nucleic acid molecule encoding, this nucleic acid molecule comprises or is made up of the nucleotide sequence of at least 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 more continuous nucleotides of SEQ ID NO:2471 nucleotide sequence or its complementary sequence.In another specific embodiments, the invention provides isolated polypeptide or albumen by nucleic acid molecule encoding, this nucleic acid molecule comprises or is made up of the nucleotide sequence of at least 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 or more continuous nucleotides of SEQ ID NO:2473 nucleotide sequence or its complementary sequence.The present invention also provides from isolating albumen of hSARS virus or polypeptide, comprises from the cellular segregation of virus infection but is not present in the viral protein of corresponding non-infected cells.The present invention also provides albumen or 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).The present invention also provides albumen or the polypeptide with SEQ ID NO:2472 or 2474 aminoacid sequences.Polypeptide of the present invention or albumen preferably have the proteic biological activity (comprising antigenicity and/or immunogenicity) of SEQ ID NO:1,11,13,2471 or 2473 sequence encodings.In other embodiments, polypeptide of the present invention or albumen have nucleotide sequence coded proteic biological activity (comprising antigenicity and/or immunogenicity), and this nucleotides sequence is classified at least 5 of SEQ ID NO:15 nucleotide sequence or its complementary sequence as, 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 more continuous nucleotides.In other embodiments, polypeptide of the present invention or albumen have Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and the proteic biological activity (comprising antigenicity and/or immunogenicity) of 12 (SEQ ID NO:1109-1589,1591-1964 and 1966-2470).The present invention also provides albumen or polypeptide, and described albumen or polypeptide have the proteic biological activity of SEQ ID NO:2472 or 2474 aminoacid sequences.

In one aspect, the invention provides the method for propagation hSARS virus in host cell, this method comprises with isolating hSARS virus infection host cell, cultivation host cell makes virus replication and results gained virosome.The present invention also provides by the host cell of hSARS virus infection.

In one aspect, the present invention relates to the purposes of isolating hSARS virus in diagnosis and methods of treatment.In a specific embodiments, the invention provides the method for using isolating hSARS virus or its any albumen or polypeptide in biological sample, to detect hSARS virus immunity specific antibody.In another embodiment, the invention provides screening immunologic opsonin combination and in and the method for the antibody of hSARS.This antibody is used for passive immunization or the immunotherapy that hSARS infects object.

The present invention also provides antibody, and described antibodies specific is in conjunction with the polypeptide of the present invention of SEQ ID NO:1,11,13,2471 or 2473 nucleotide sequences or its fragment coding or be included under the stringent condition with the polypeptide of the present invention of the nucleic acid encoding of the nucleotide sequence of SEQ ID NO:1,11,13,2471 or 2473 nucleotide sequence hybridizations and/or have the bioactive any hSARS epi-position of one or more polypeptide of the present invention.The present invention also provides the antibody of specificity in conjunction with the polypeptide of the present invention of SEQID NO:15 nucleotide sequence or its fragment coding.These polypeptide comprise the polypeptide shown in Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and 12 (SEQ IDNO:1109-1589,1591-1964 and the 1966-2470).In another embodiment, this polypeptide comprises SEQ ID NO:2472 or 2474 aminoacid sequences.The present invention also provides antibody, described antibodies specific is in conjunction with by the polypeptide of the present invention that is included under the stringent condition with the nucleic acid encoding of the nucleotide sequence of SEQ IDNO:15 nucleotide sequence hybridization, and/or has the 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 ') ₂Fvs, the intracellular antibody that fragment, disulfide linkage connect and contain specificity in conjunction with the VL of polypeptide of the present invention or VH territory or even the fragment of complementary determining region (CDR).

In another embodiment, the invention provides the vaccine product of the protein subunit that comprises hSARS virus (reorganization and the chimeric form that comprise described virus) or described virus.In a specific embodiments, vaccine product of the present invention comprises alive but the hSARS virus that is with or without adjuvant of attenuation.In another embodiment, vaccine product of the present invention comprises deactivation or dead hSARS virus.Can go down to posterity or virus by preparation reorganization or chimeric form prepares this attenuation or inactivation of viruses by virus a series of in host cell.Therefore, the present invention also provides the method for preparing reorganization or chimeric form hSARS.In another embodiment, vaccine product of the present invention comprises the nucleic acid or the fragment of hSARS virus (for example searching number is the virus of CCTCC-V200303), or its sequence is SEQ ID NO:1,11,13,15,2471 or 2473 or its segmental nucleic acid molecule.In another embodiment, the invention provides the vaccine product that comprises one or more polypeptide isolating or that produce from the hSARS viral nucleic acid from hSARS virus, described virus for example preservation searching number is the virus of CCTCC-V200303.In a specific embodiments, vaccine product comprises the polypeptide of the present invention of SEQID NO:1,11,13,2471 or 2473 nucleotide sequences or its fragment coding.In a specific embodiments, vaccine product comprises the polypeptide of the present invention described 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) or the polypeptide of the present invention of SEQ ID NO:15 nucleotide sequence or its fragment coding.In a specific embodiments, vaccine product comprises the polypeptide that contains SEQ ID NO:2472 or 2474 aminoacid sequences.In addition, the invention provides by giving separately or can accepting vehicle with adjuvant or other medicines and unite the method that SARS was treated, improves, controls or prevented to the vaccine product of the present invention or the antibody of use.

In yet another aspect, the invention provides the pharmaceutical composition that comprises antiviral agent of the present invention and drug acceptable carrier.In a specific embodiments, antiviral agent of the present invention is the antibody of immunologic opsonin in conjunction with hSARS virus or any hSARS epi-position.In preferred embodiments, in this antibody and hSARS virus.In a specific embodiments, antiviral agent of the present invention is in conjunction with fragment, variant, the homologue of hSARS virus N-gene or S-gene.In a specific embodiments, antiviral agent of the present invention is in conjunction with the fragment, variant, the homologue that comprise the polypeptide of SEQ ID NO:2472 or 2474 aminoacid sequences.In another embodiment, antiviral agent is polypeptide of the present invention or albumen or nucleic acid molecule of the present invention.The present invention also provides the medicine box that comprises pharmaceutical composition of the present invention.

3.1 definition

Term used herein " immunologic opsonin is in conjunction with the antibody or the antibody fragment of polypeptide of the present invention " refers to immunologic opsonin in conjunction with polypeptide or its fragment shown in SEQ ID NO:1,11,13,15,2471 or 2473 nucleotide sequence coded polypeptide or Figure 11 or 12, and not non-specific antibody or its fragment in conjunction with other polypeptide.Immunologic opsonin in conjunction with the antibody of polypeptide of the present invention or its fragment can with other antigen generation cross reaction.Preferred immunologic opsonin in conjunction with the antibody of polypeptide of the present invention or its fragment not with other antigen generation cross reaction.Immunologic opsonin can be identified by for example immunoassay or other technology well known to those skilled in the art in conjunction with antibody or its fragment of polypeptide of the present invention.

" isolating " or " purifying " peptide or protein do not contain substantially from the cell material of described proteinic cell source or tissue source 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 component 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, the volume of promptly cultivating the fiduciary point protein articles is less than about 20%, 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 participate in protein synthesis.Therefore, this polypeptides goods contain and are less than about 30%, 20%, 10%, 5% (dry weight) precursor or the compound that are not desired polypeptides/protein fragments.In the preferred embodiment of the invention, polypeptides is isolating or purifying.

" 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 such as 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 the preferred embodiment of the invention, the nucleic acid molecule of code book invention polypeptides is isolating or purifying.Term " isolating " nucleic acid molecule does not comprise not and the isolating library of other library clone member's nucleic acid that contains other nucleic acid molecule.

Term used herein " part " or " fragment " refer to that the length that contains the associated nucleic acid molecule is at least about 25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1,000,1,050,1,100,1,150,1,200,2,000,3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,11,000,12,000,13,000,14,000,15,000,16,000,17,000,18,000,19,000,20,000,21,000,22,000,23,000,24,000,25,000,26,000,27,000,28,000,29,000 or more a plurality of continuous nucleic acid, and have the nucleic acid molecule fragment of the functional character (or its encoded protein matter has the coded proteinic a kind of functional character of associated nucleic acid molecule) of at least a associated nucleic acid molecule; Or refer to contain at least 5 of related protein or polypeptide, 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,90,100,120,140,160,180,200,220,240,260,280,300,320,340,360,400,500,600,700,800,900,1,000,1,500,2,000,2,500,3,000,3,500,4,000,4,100,4,200,4,300,4,350,4,360,4,370,4,380 amino-acid residue length, and have the protein or the polypeptide fragment of the functional character of at least a related protein or polypeptide.

Term " the virus genomic 3 ' district of hSAR " refers to about 18000-29742 position Nucleotide of SEQ ID NO:15.

Term " has the proteinic biological activity of the present invention " or " biological activity with polypeptide of the present invention " refers to have common bioactive polypeptide or proteinic characteristic, it is compared with SEQ IDNO:1,11,13,15,16,240,737,1108,1590,1965,2471 or 2473 nucleotide sequence coded polypeptide, has similar or same structure territory and/or have enough amino acid consistence.This common biological activity of polypeptide of the present invention comprises antigenicity and immunogenicity.

Term " under stringent condition " refers to have each other at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% conforming nucleotide sequence and keeps the hybridization and the wash conditions of hybridization mutually.This hybridization conditions is such as but not limited at Current Protocols in Molecular Biology, John Wiley﹠amp; Sons, N.Y. (1989), 6.3.1-6.3.6.; Basic Methods in Molecular Biology, ElsevierScience Publishing Co., Inc., N.Y. (1986), 75-78 page or leaf and 84-87 page or leaf; And Molecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982) describes in the 387-389 page or leaf, knows for a person skilled in the art.Preferably the limiting examples of stringent hybridization condition is, hybridizes among 6 * sodium chloride/sodium citrate (SSC), the 0.5%SDS down at about 68 ℃, at room temperature washs one or many among 2 * SSC, the 0.5%SDS then.The limiting examples of another preferred stringent hybridization condition is, hybridizes among 6 * SSC down at about 45 ℃, washs one or many then in about 50-65 ℃ time 0.2 * SSC, 0.1%SDS.

Term used herein " variant " refers to the hSARS mutant of the hSARS transgenation strain or the reorganization preparation of natural generation, compares with the hSARS of CCTCC-V200303, contains one or more sudden changes in their each comfortable its genomes.Term " variant " also can refer to the mutant of natural generation of particular peptide or the mutant of particular peptide or proteinic reorganization preparation, and wherein one or more amino-acid residues are modified by amino-acid substitution, insertion or disappearance.

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-specific 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 the ultracentrifugal sedimentation thing of the hSARS virus of pH 7.0 usefulness 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 cells infected.

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 SARS 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 SARS virus acquisition and the aminoacid sequence (SEQ ID NO:14) of inferring thereof.

Figure 10 shows the whole genome dna sequence dna (SEQ ID NO:15) of SARS 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 complementary sequence 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 N-gene primer sequence (it is at the 29247-29410 position of SEQ ID NO:2471 amplification of nucleotide); 150# (SEQ ID NO:2475); 200# (SEQ ID NO:2476); And S-gene primer sequence (it is at the 24751-25049 position of SEQ ID NO:2473 amplification of nucleotide); 131# (SEQ ID NO:2477); 132# (SEQ ID NO:2478).

Figure 14 A shows the nucleotide sequence (SEQ ID NO:2471) of N-gene.Figure 14 B shows the aminoacid sequence (SEQ ID NO:2472) of N-gene.

Figure 15 A shows the nucleotide sequence (SEQ ID NO:2473) of S-gene.Figure 15 B shows the aminoacid sequence (SEQ ID NO:2474) of S-gene.

Figure 16 shows the genome organization of SARS-CoV HK-39 and transcribes strategy.Genome and mRNA transcript are added cap (black circle), carry leader sequence (vertical line) at 5 ' near-end, and by poly-adenosineization (A15).Arrow refer to intergenic sequence 5 '-position of CTAAACGAAC-3 ' (SEQ IDNO:2479).After just geneome RNA is discharged in the host cell tenuigenin, the viral RNA-RNA-dependent polysaccharase of synthetic ORF 1a and 1b coding.It carries out transcribing of total length complementation (antisense) RNA, from the synthetic new geneome RNA of this RNA, the contig (overlapping set) and the leading RNA of subgenomic mRNA transcript.It should be noted that before all transcripts common 5 ' leader sequence and common 3 ' end are all arranged.ORF1a and 1b-RNA-RNA-dependent polysaccharase; The peplomerbody glycoprotein that S-is main; The M-transmembrane glycoprotein; The N-nucleocapsid; The albumen that X1, X2, X3-infer.

Figure 17 shows the design of graphics of pSARSCoV-ORF1b-N.Be connected to jointly the cloning vector pCR2.1-TOPO (Invitrogen) from the ORF1b (1b) of SARS-CoV and the PCR product of N gene amplification.Nucleotide (nt) number is equivalent to the position in the sequence of HK-39 strain SARS-CoV (AY278491).The amplicon of primer (being SEQ ID NO:2480 and the 2481) amplification of diagnostic assay is represented to be used in the shadow region.

Figure 18 shows the sepharose photo behind the total RNA electrophoresis that extracts from SARS patient with the total RNA separation system of SV.The RNA that extracts tests to detect the coronavirus among the patient through RT-polymerase chain reaction (RT-PCR) then.

Figure 19 shows the influence of potential inhibitor in the RT-polymerase chain reaction (RT-PCR).For removing potential inhibitor, with 95% ethanol and 3M sodium acetate precipitation, be resuspended in the water of 12 μ l nuclease free from total RNA of SV96 board wash-out.Carry out RT-PCR with actin-F (SEQ ID NO:2482) and actin-R (SEQ ID NO:2483) primer.The numeral that shows is the quantity that adds the pig renal epithelial cell (PK-15) in the sample as internal contrast.The RNA sample does not amplify dna fragmentation with being untreated.

Figure 20 shows the primer of the range gene that is used to increase.SRS251 (SEQ ID NO:2480) and SRS252 (SEQ ID NO:2481) amplify the fragment of 225 base pairs from the N-gene regions, and this district and other coronavirus do not show homology.Coro3 (SEQ ID NO:3) and coro4 (SEQ BD NO:4) amplification RNA-RNA-dependent polysaccharase (1b gene) in contrast.Actin-F (SEQ ID NO:2482) and actin-R (SEQ ID NO:2483) go out the fragment of 745 base pairs from the beta-actin gene amplification as PCR test internal contrast.

Figure 21 A shows the amplification figure that fluorescence intensity is done the PCR cycle number.Black line is represented the dynamicrange of N-gene specific PCR, and the plasmid construction body of its serial dilution is the 101-106 copy.Non-SARS patient, comprise infect adenovirus (n=5), respiratory syncytial virus (n=5), human stroma lung virus (human metapneumovirus) (n=5), the patient's of influenza A virus (n=5) or influenza B virus (n=5) NPA sample represents with gray line.The line of band triangle is represented the positive NPA sample of SARS-CoV; NTC represents not have the template contrast; The X-axle is represented the cycle number of the quantitative PCR that carries out, and the Y-axle represents to surpass the fluorescence intensity (FAM-400) (Δ Rn) of background signal.Insert the curve analysis of figure expression PCR product.Shown from positive (+ve) sample, the feminine gender (ve) signal that contrasts of sample and no template.The X-axle represent temperature (℃), and the Y-axle is represented fluorescence intensity (Δ Rn).The comparison of the dynamicrange of the specific PCR of Figure 21 B demonstration N-gene and 1b-gene.The dynamicrange of N-gene and 1b-gene PCR all obtains with identical plasmid construction body, and wherein corresponding amplicon was with 1: 1 ratio subclone.Copy number is that the plasmid of the serial dilution of 10-1-105 copy is used as the template among two PCR.The line of band triangle is represented N-gene specific PCR, and gray line is represented 1b-gene specific PCR.Ct value ± standard deviation when inserting figure expression use different templates copy number in three repetitions of two PCR experiments.NTC represents not have the template contrast; The X-axle is represented the cycle number of the quantitative PCR that carries out, and the Y-axle is represented fluorescence intensity.

Figure 22 A and 22B show amplification curve and the melting curve to the 1b of SARS CoV (use has the primer of SEQ IDNO:3 and 4) and the specific real-time quantitative PCR of N-gene (use has the primer of SEQ ID NO:2480 and 2481) respectively.Figure 22 A: fluorescence intensity is made amplification figure to the PCR cycle number.NPA, the tracheae of having the patient of clinical symptom by oneself disperse the template of 1 μ l cDNA of thing (tracheal dispersion) and lung biopsy as each PCR.PCR carries out 50 circulations to reach the period of saturation of reaction.The X-axle is represented the cycle number of the quantitative PCR that carries out, and the Y-axle represents to surpass the fluorescence intensity (FAM-490) of background signal.Horizontal gray line is represented to calculate baseline circulation Ct automatically by the threshold value of maximum curvature method calculating.Insertion figure represents to use half maximum fluorescence value (1/2 maximum) and the Ct of two PCR of the cDNA that derives from various tissues, these are organized in three different time points and separate from crucial patient (the patient A that is mentioned among the New Engl.J.Med.348:1967-76 (Drosten etc., 2003)).NPA=nasopharynx aspirate; TW=tracheae washes; LW=lung washes.The melting curve of Figure 22 B:PCR product.After further extending step, 10 minutes reactions carry out the analysis of melting curve.Temperature is divided and is elevated to 94 ℃ from 56 ℃ 76 times, each 0.5 ℃ of increment, and the temperature of each setting point keeps 7 seconds to collect data and analysis.The melting temperature(Tm) of 1b-and N-gene specific PCR product is respectively 80.5 ℃ and 85.5 ℃.The X-axle is the temperature with degree centigrade expression, and the Y-axle represents to surpass the fluorescence intensity (FAM-490) of background signal.1 μ l water is used as no template contrast in reaction.

Figure 23 shows the diagnostic result of 48 clinical samples, wherein uses the primer with SEQ IDNO:2480 and 2481 respectively, with beta-actin PCR as internal contrast.The band of each top, road is represented the 745bp dna fragmentation with actin-F and actin-R amplification, and the band of below is the amplicon of sars coronavirus (225bp) N-gene-specific primer.Represented in the test-ve contrast (water) and+ve contrast (from the cDNA of the Vero cell of sars coronavirus infection).Mix 5 μ l PCR products of two reactions, be added in the sample well of 2% sepharose.M=1kb+ molecular weight marker (Invitrogen).

Figure 24 shows the rna blot analysis of the total RNA of SARS-CoV.Total RNA of the Vero E6 cell extraction SARS-CoV that infects from SARS-CoV.RNA separates in containing 1% denaturant gel of 6.29% formaldehyde.RNA transfers on the positive polarity nylon membrane then, hybridizes with the digoxigenin labeled fragment to 1b, S, M and N gene specific respectively.1 road-1b; 2 roads-S; 3 roads-M; 4 roads-N.Vertical bar is represented the molecular weight reference.Arrow is represented the transcript with the N probe hybridization.Signal is by chemiluminescence analysis.

Figure 25 shows the dna probe that is used for rna blot analysis.Represented 1b gene (Nucleotide 18057-18222; SEQ ID NO:2484), S gene (Nucleotide 21920-22107; SEQ ID NO:2485), M gene (Nucleotide 25867-26996; SEQ ID NO:2486) and N gene (Nucleotide 28658-28883; SEQ ID NO:2487) probe.

                     5. detailed Description Of The Invention

The inventor has developed quick, the high flux reverse transcription-pcr diagnostic test method that is used for SARS associated coronavirus (SARS-CoV). Comprise that the hSARS viral genome 3 of nucleocapsid gene (N-gene) ' district shows as responsive molecular labeling, can be beyond the 1b gene for increasing the sensitiveness of test. Use the PK-15 cell can in order to guarantee the integrality of RNA when leaching process and cDNA are synthetic, therefore eliminate false negative result as internal contrast.

In the prototypical member MHV (MHV) of coronavirus genus, geneome RNA and mRNA transcript are added cap, have 3 common ' end and common 5 ' end targeting sequencing. According to the strategy of transcribing of this uniqueness, the copy number difference (Figure 19) of virus different virus gene when host's internal breeding. The N gene of coding nucleocapsid has the abundantest copy number during virus replication, because all transcript portabilities are from the nucleotide sequence of N gene, although they are not all in translating the frame of this gene outcome. The inventor has found the diagnostic test of (comprising the N-gene) based on viral genome 3 ' district, and the test more responsive than viral genome remainder is provided. Therefore, in preferred embodiments, the nucleic acid molecules that can be used for diagnostic test comprises nucleotide sequence or its part of the 18000-29742 position nucleotides of SEQ ID NO:15. This part can comprise 15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,750,800,850,900,950,1000,1050,1100,1150,1200 nucleotides that have from the nucleotide sequence of the 18000-29742 position nucleotides of SEQ ID NO:15. In other preferred embodiment, the nucleic acid molecules that is used for diagnostic test comprises the 28658-28883 of SEQ ID NO:15 or the nucleotide sequence of 29247-29410 position nucleotides.

Obtain nasopharynx aspirate (NPA) and excrement sample from the SARS suspected patient with main clinic symptoms and obvious close contact infected patient history. From the total RNA of patient's sample extraction, simultaneously with the PK-15 cell as internal contrast. By reverse transcription PCR analysis of experiments sample. Carry out the inferior genome transcript of rna blot analysis to show that virus is different. Use real-time quantitative PCR relatively to be used for the sensitiveness at two kinds of positions of this diagnostic test. In a specific embodiments, behind the RNA leaching process, remove the PCR inhibitor with precipitation with alcohol.

In preferred embodiments, the invention provides the method that whether detection N-gene nucleic acid exists in biological sample. The method comprises from various sources acquisition biological samples, and sample is contacted with compound or the reagent that can detect hSARS virus N-gene nucleic acid (for example mRNA, geneome RNA), thus the existence of N-gene in the test sample. In preferred embodiments, the N-gene can be with the labeling nucleic acid probe in detecting that comprises SEQ ID NO:2471 nucleotide sequence or its complementary series or its part. This partial-length can be 10,20,30,40,50,100,200,400,500,600,800,1000,1200 nucleotides. In preferred embodiments, comprise SEQ ID NO:2475 and/or 2476 or the primer of SEQ ID NO:2480 and/or 2481 nucleotide sequences can be used for increasing the part of N-gene to detect.

The reagent that is preferred for detecting hSARS mRNA of the present invention or geneome RNA for can with the mRNA of code book invention polypeptide or the labeling nucleic acid probe of geneome RNA hybridization. This nucleic acid probe for example can be the nucleic acid molecules that comprises or be made up of nucleotide sequence, described nucleotides sequence is classified SEQ ID NO:1,11,13,15,2471 or 2473 or its complementary series or its part as, for example length is at least 15,20,25,30,50,100,250,500,750,1000 or more continuous nucleotides, and is enough under stringent condition the oligonucleotides with hSARS mRNA or geneome RNA specific hybrid.

In another preferred specific embodiments, use is based on the genome nucleotide sequence of N-Gene Partial nucleotide sequence or SEQ ID NO:15 or the primer that makes up based on SEQ ID NO:1,11,13,15,2471 or 2473 nucleotide sequences, by the existence of N-gene in RT-polymerase chain reaction (RT-PCR) test sample. In a non-limiting specific embodiments, the preferred primer that uses is in the RT-PCR method: 5 '-TACACACCTCAG-CGTTG-3 ' (SEQ IDNO:3) and/or 5 '-CACGAACGTGACGAAT-3 ' (SEQ ID NO:4), at 2.5mM MgCl₂Exist lower, thermal cycle for example for but be not limited to 94 ℃ 8 minutes, then 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ circulations of 1 minute 40 times (also referring to following 6.7 and 6.8 joints). In preferred embodiments, primer comprises SEQ ID NO:2475 and 2476 nucleotide sequences. In a further preferred embodiment, primer comprises SEQ ID NO:2480 and 2481 nucleotide sequences. In preferred embodiments, thermal cycle be 94 ℃ 10 minutes, 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ circulations of 30 seconds are 40 times then, 72 ℃ 10 minutes. In a further preferred embodiment, thermal cycle be 94 ℃ 3 minutes, 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ circulations of 30 seconds are 40 times then, 72 ℃ 10 minutes. In how preferred specific embodiments, the invention provides real-time quantitative PCR and test existing with hSARS virus in the detection of biological sample, by making from the total RNA reverse transcription of the extraction of sample, with the cDNA that obtains with Auele Specific Primer (primer that for example has SEQ ID NO:3 and 4 nucleotide sequences) and fluorescent dye (SYBR for example^_Green I, it fluoresces when the non-specific binding double-stranded DNA) carry out the PCR reaction. Because the thermal cycle through some has produced the PCR product, when prolonging the end of step, catch the fluorescence signal of these reactions, therefore can be based on virus quantity (referring to following 6.7) in the amplification figure quantitative assay sample.

In preferred embodiments, the invention provides the method that whether detection S-gene nucleic acid exists in biological sample. The method comprises from various sources acquisition biological samples, and sample is contacted with compound or the reagent that can detect hSARS virus S-gene nucleic acid (for example mRNA, geneome RNA), thus the existence of S-gene in the test sample. The reagent that is preferred for detecting hSARS mRNA of the present invention or geneome RNA for can with the mRNA of code book invention polypeptide or the labeling nucleic acid probe of geneome RNA hybridization. This nucleic acid probe for example can be the nucleic acid molecules that comprises or be made up of SEQ ID NO:1,11,13,15,2471 or 2473 nucleotide sequences or its complementary series or its part, for example length is at least 15,20,25,30,50,100,250,500,750,1000 or more continuous nucleotides, and is enough under stringent condition the oligonucleotides with hSARS mRNA or geneome RNA specific hybrid.

In another preferred specific embodiments, use the primer that makes up based on S-Gene Partial nucleotide sequence (SEQ ID NO:2473), by the existence of S-gene in RT-polymerase chain reaction (RT-PCR) test sample.

External technology for detection of mRNA comprises Northern hybridization, in situ hybridization, RT-PCR and RNA enzyme protection. External technology for detection of geneome RNA comprises Nothern hybridization, RT-PCT and RNA enzyme protection.

The polynucleotides of coding N-gene 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, T4 archaeal dna 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. 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 at 70 ℃.

The primer that is used for amplification N-gene of the present invention or S-gene 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 N-gene of the present invention or S-gene code polynucleotides in conjunction with the disclosure.

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 (OLAs) (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, the DNA sample that contains on a small quantity the polynucleotides that obtain from tissue or patient 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 inventive method can comprise real-time quantitative PCR test, for example Taqman^_Test (Holland etc., Proc Natl Acad Sci USA, 88 (16): 7276 (1991); Also referring to the U.S. Patent application of submitting on March 24th, 2004, attorney docket V9661.0078, its by reference integral body be attached to herein). The present invention test can be carried out being designed for the instrument that carries out this test, for example can be available from the instrument of Applied Biosystems (Foster City, CA). The primer and the probe that are used for this test can design according to method known to those skilled in the art.

The length that is used for the primer of amplification N-gene or S-Gene Partial is at least 10,15,20,25,30 nucleotides. Specifically, most preferably the increase primer of N-gene or S-gene. 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 40,60,100,200,300,400,500,600,800,1000 base-pairs.

In a specific embodiments, the inventive method further relates to from contrast object acquisition control sample, control sample is contacted with compound or the reagent that can detect N-gene or S-gene, thereby coding N-gene or the mRNA of S-gene or the existence of geneome RNA in the test sample, and with the mRNA of N-gene in the control sample or S-gene or coded polypeptide or geneome RNA whether exist with testing sample in the mRNA of N-gene or S-gene or coded polypeptide or geneome RNA exist and compare.

The present invention also is included in and detects the kit that the N-gene nucleic acid exists in the testing sample. This kit, for example, can comprise labeled compound or the reagent that can in testing sample, detect polypeptide encoding nucleic acid molecule, and the method (in conjunction with the DNA of coded polypeptide or the oligonucleotide probe of mRNA) that mRNA measures in the test sample in some embodiment.

For the kit based on oligonucleotides, this kit can for example comprise: (1) oligonucleotides, detectable labeled oligonucleotide for example, nucleotide sequence or the intragenic sequence hybridization of N-of itself and code book invention polypeptide; (2) be used for the primer pair that amplification contains the nucleic acid molecules of N-gene order. 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 essential 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 loaded into independent container usually, and all various containers and operation instruction are enclosed in the individual packaging thing together.

The present invention relates to N-gene and S-gene that hSARS virus is separated. In a specific embodiments, described virus comprises SEQ ID NO:1,11,13,15,2471 and/or 2473 nucleotide sequences. In a specific embodiments, the invention provides the isolated nucleic acid molecule of hSARS virus, this nucleic acid molecules comprises or is made up of SEQ ID NO:1,11,13,15,2471 and/or 2473 nucleotide sequences or its complementary series or its part. In another embodiment, the invention provides under the stringent condition of this paper definition and nucleic acid molecules or the known member's of coronaviridae specific gene or the isolated nucleic acid molecule of its complementary sequence hybridization with SEQ ID NO:1,11,13,15,2471 and/or 2473 sequences. 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 complementary series 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 ID NO:11 nucleotide sequence or its complementary series at least about 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,700,750,800,850,900,950,1,000,1,050,1,100,1,150,1,200 or the nucleotide sequence of more continuous nucleotides. In another specific embodiment, 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 complementary series 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 the nucleotide sequence of at least 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 more continuous nucleotides of SEQ ID NO:2471 nucleotide sequence or its complementary series. In another specific embodiments, the invention provides isolated polypeptide or the protein of nucleic acid molecule encoding, described nucleic acid molecules comprises the nucleotide sequence of at least 5,10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1,100,1150,1200,2000,3000 or more continuous nucleotides of SEQ ID NO:2473 nucleotide sequence or its complementary series. In another specific embodiment, the invention provides isolated polypeptide or protein by nucleic acid molecule encoding, described nucleic acid molecules comprises at least 5 of SEQ ID NO:15 nucleotide sequence or its complementary series, 10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1,000,1,050,1,100,1,150,1,200,2,000,3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,11,000,12,000,13,000,14,000,15,000,16,000,17,000,18,000,19,000,20,000,21,000,22,000,23,000,24,000,25,000,26,000,27,000,28,000,29,000 or the nucleotide sequence of more continuous nucleotides. These polypeptide comprise the polypeptide shown in Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964 and 1966-2470) or have SEQ ID NO:2472 or the polypeptide of 2474 amino acid sequences. Polypeptide of the present invention or protein preferably have one or more biologically actives of following protein: by the protein of SEQ ID NO:1,11,13,15,2471 or 2473 sequential codings; Or the polypeptide shown in Figure 11 and Figure 12; Or contain natural viral protein by the amino acid sequence of SEQ ID NO:1,11,13,15,2471 or 2473 sequential codings.

The invention still further relates to the method for propagation hSARS virus in host cell.

The invention further relates to the application of sequence information in diagnostic method and methods of treatment of isolated viral.In a specific embodiments, the invention provides whole nucleotide sequence SEQ ID NO:15 or its fragment or the complementary sequence of hSARS virus CCTCC-V200303.In addition, the present invention relates to can be under stringent condition and the nucleic acid molecule of any part hybridization of the genome SEQ ID NO:15 of hSARS virus CCTCC-V200303.In a specific embodiments, the invention provides the nucleic acid molecule that is suitable as primer, this nucleic acid molecule comprises or is made up of SEQ ID NO:1,11,13,15,2471 or 2473 nucleotide sequences or its complementary sequence or its part.In a specific embodiments, described primer comprises SEQID NO:2475,2476,2477,2478,2480 or 2481 nucleotide sequences.In another embodiment, the invention provides the nucleic acid molecule that is suitable as hybridization probe, be used to detect and comprise or by SEQ ID NO:1,11,13,15,2471 or 2473 nucleotide sequences or its complementary sequence or its part nucleic acid that form, code book invention polypeptide.The invention still further relates to the test kit that comprises primer, described primer has SEQ ID NO:2475 and 2476 and SEQ ID NO:2480 and 2481 nucleotide sequences, to survey the N-gene.In another embodiment, the present invention relates to comprise the test kit of primer, described primer has SEQ ID NO:2477 and/or 2478 nucleotide sequences, to survey the S-gene.In a preferred embodiment, described test kit also comprises reagent, is used for detecting the non-existent gene of hSARS virus as negative control.The present invention further comprises embedded virus or recombinant virus or by described nucleotide sequence coded virus protein.

The present invention further provides the combination of energy specificity by the polypeptide of the present invention of SEQ ID NO:1,11,13,2471 or 2473 nucleotide sequences or its fragment coding or the antibody of any hSARS epi-position.It is the antibody of the polypeptide of SEQ ID NO:2472 or 2474 in conjunction with its aminoacid sequence that the present invention also provides specificity.The present invention further provides specificity in conjunction with antibody by polypeptide shown in the polypeptide of the present invention of SEQ ID NO:15 nucleotide sequence or its fragment coding or Figure 11 and 12 or its fragment or any hSARS epi-position.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 ') ₂Fvs, the intracellular antibody that fragment, disulfide linkage connect and contain can with polypeptid specificity bonded VL of the present invention or VH structural domain or even the fragment of complementary determining region (CDR).

In one embodiment, the invention provides the method for the existence, activity or the expression that in biomaterial such as cell, blood, saliva, urine, phlegm, nasopharynx aspirate etc., detect hSARS virus of the present invention.Can be by biomaterial and the reagent that can directly or indirectly detect the existence of hSARS virus being contacted to determine the existence of hSARS virus in the sample.In a specific embodiments, detection reagent is an antibody of the present invention.In another embodiment, detection reagent is a nucleic acid molecule of the present invention.

In another embodiment, the invention provides the vaccine product that comprises hSARS virus (recombinant forms and the chimeric form that comprise described virus) 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 vehicle, 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 or.Therefore, the present invention further provides the recombinant forms of preparation hSARS or the method for chimeric form.In another embodiment, vaccine product of the present invention comprises one or more nucleic acid molecule that comprises or be made up of SEQ IDNO:1,11,13,15,2471 and/or 2473 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:1,11,13,2471 and/or 2473 nucleotide sequences or its fragment form nucleotide sequence coded, or be polypeptide shown in Figure 11 and 12 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: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: antiviral agent [amantadine for example, Rimantadine, ganciclovir, acyclovir, ribavirin, Penciclovir, oseltamivir, phosphine formic acid, zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), nevirapine, delavirdine, Indinavir, ritonavir, vidarabine, viracept see nelfinaivr, Saquinavir, zanamivir, oseltamivir phosphate, pleconaril, Interferon, rabbit etc.], steroid and reflunomide are (as prednisone, cortisone, fluticasone) and glucocorticosteroid, microbiotic, pain killer, bronchodilator or other are used for the therapy of respiratory tract and/or virus infection.

In addition, the invention provides the pharmaceutical composition that comprises antiviral agent 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 the infectivity of hSARS virus or its variant or the method for the antiviral agent that duplicates.

5.1 reorganization and chimeric hSARS virus

The present invention includes origin and come from reorganization or the embedded virus that hSARS genomic virus vector viral or its natural variant is encoded.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 has the different sequence of genome sequence (SEQ ID NO: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 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 ID NO:1,11,13,2471,2473 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:2,12,14,2472 or 2474 aminoacid sequence or its fragment as shown in Figure 11 (SEQID NO:17-239,241-736 and 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964 and 1966-2470) 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 SARS 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 (1998); 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 as other virus, use attenuation and replication-defective 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, so that the antigen sequence that the infection of hSARS and natural variant thereof is brought provide protection to be provided.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 body can advantageously be applied in the vaccine product.Can carry out artificial reconstructedly to expression product of the present invention and embedded virus body, 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 variant infect to make to embedded virus body of the present invention.

In certain embodiments, can carry out artificial reconstructedly to expression product of the present invention and reorganization or embedded virus body, 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, and described virus vector derives from hSARS virus or its variant, 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.Sudden change 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 or insertion to produce attenuated virus.In addition, the invention provides the host cell that infects hSARS virus (for example preserving number is the hSARS virus of CCTCC-V200303).

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.

5.2 the preparation of 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, the polypeptide with SEQ ID NO:2472 or 2474 aminoacid sequences or its antigen fragment shown in SEQ IDNO:1,11,13,15,2471,2473 nucleotide sequence coded polypeptide, Figure 11 (SEQID NO:17-239,241-736 and 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964 and 1966-2470), 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; certainly 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.

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.

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.

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 nucleic acid molecule or its fragment of SEQ ID NO:1,11,13,15,2471,2473 sequences.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 links to each other with one or more controlling elementss effectively, thereby makes and can be expressed in the 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 virus 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 haemaglutinin-expressing 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.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, Direct gene transfer intomouse muscle in vivo, Science 247:1465-1468; Raz, E., 1994, Intradermal gene immunization:The possible role of DNA uptake in theinduction 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 eliciting an immuneresponse, Nature 356:152-154).The comprehensive review of the method for relative dna vaccine is referring to Robinson, H.L., and 1999, DNA vaccines:basic mechanism and immuneresponses (summary), Int.J.Mol.Med 4 (5): 549-555; Barber, B., 1997, Introduction:Emerging vaccine strategies, Seminars in Immunology9 (5): 269-270; And Robinson, H.L. etc., 1997, DNA vaccines, Seminarsin Immunology 9 (5): 271-283.

5.3 the attenuation of hSARS virus or its variant

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 virion.

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.

Can carry out attenuation to virus of the present invention, so that one or more functional characters of virus are impaired.In certain embodiments, 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, can duplicate in the host, thereby produce infectious virion.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 (for example reorganization or chimeric hSARS) in people's cell, duplicate not as wild-type virus (for example wild-type hSARS) 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 virion.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 is that attenuation hSARS can not duplicate 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 is introduced in SEQ ID NO:1,11,13,15,2471 or 2473 sequences, has 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.Sudden change can be to increase, replace, lack or their combination.Can screen the expectation function of this hSARS variant, 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, 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 virosomal membrane.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 virosome.If heterologous protein is inserted in the virosome, then should detect virosome causes symptom in appropriate animal model ability, because virus may obtain new, possible morbific character.

5.4 adjuvant and carrier molecule

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 (cochleates) 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 (CTB), 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 carboxy 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.

Vaccine of the present invention can be polyvalent vaccine or univalent vaccine.Polyvalent vaccine is made by instructing more than a kind of recombinant virus of antigen presentation.

Can use several different methods to introduce vaccine product of the present invention; That these methods include but not limited to is oral, approach and by scratch method (for example using minute forked needle to scratch the upper strata of skin) in intracutaneous, intramuscular, intraperitoneal, intravenously, subcutaneous, the nose.

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

5.5 the preparation of antibody

Specific recognition polypeptide of the present invention, such as but not limited to comprising the polypeptide shown in SEQ ID NO:2,12,14,2472,2474 polypeptide of sequence and Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964,1966-2470), or the antibody of hSARS epi-position or its Fab, 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 polypeptide of the present invention or preferred hSARS in sample biological example material, described biomaterial comprises cell, cell culture medium (for example bacterial cell substratum, mammalian cell substratum, insect cell substratum, yeast cell substratum etc.), blood, blood plasma, serum, tissue, phlegm, nasopharynx aspirate etc.

Any epi-position of polypeptide of the present invention or hSARS 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 aluminium hydroxide, surfactant such as lysolecithin, polyether glycol, polyanion, peptide, fat liquor, keyhole _ hemocyanin, dinitrophenol and the mankind is 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 display technique of bacteriophage 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 in can strengthening or reduce the bioactive any part of antibody, introduce 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 the identical selective marker expression of heavy chain and light chain polypeptide (make equate) 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, to avoid occurring excessive poisonous free heavy chain (Proudfoot, Nature, 322:52,1986 and Kohler, Proc.Natl.Acad.Sci.USA, 77:2197,1980).The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna.

In another embodiment, antibody also can use various phage display method well known in the art to produce.In the phage display method, the function antibody structural domain is illustrated on the surface of the phage particle that carries its coded polynucleotide sequence.In a specific embodiments, this phage can be used to show 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 the recombinant fusion protein that merges with phage gene III or gene VIII protein.The example that can be used to prepare immunoglobulin (Ig) of the present invention or its segmental phage display method is included in disclosed method in the following document: Brinkman etc., J.Immunol.Methods, 182:41-50,1995; Ames etc., J.Immunol.Methods, 184:177-186,1995; Kettleborough etc., Eur.J.Immunol., 24:952-958,1994; Persic etc., Gene, 187:9-18,1997; Burton etc., Advances in Immunology, 57:191-280,1994; PCT application number PCT/GB91/01134; PCT publication number WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; 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 complete antibody, comprise people's antibody, or any fragment that 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., BioTechniques, 12 (6): 864-869,1992; And Sawai etc., AJRI, 34:26-34,1995; And Better etc., Science, 240:1041-1043,1988 (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., Methods in Enzymology, 203:46-88,1991; Shu etc., PNAS, 90:7995-7999,1993; And Skerra etc., Science, 240:1038-1040,1988.

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 albumen and the G protein purification 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, Science, 229:1202,1985; Oi etc., BioTechniques, 4:214 1986; Gillies etc., J.Immunol.Methods, 125:191-202,1989; 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 combination of advantageous embodiment antigen.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 rare framework residue at specific position.Referring to for example Queen etc., U.S. Patent number 5,585,089; Riechmann etc., Nature, 332:323,1988, above document integral body by reference is attached to herein.Antibody can use multiple 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, MolecularImmunology, 28 (4/5): 489-498,1991; Studnicka etc., ProteinEngineering, 7 (6): 805-814,1994; Roguska etc., Proc Natl.Acad.Sci.USA, 91:969-973,1994) 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 display technique of bacteriophage.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, Int.Rev.Immunol., 13:65-93,1995.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 0 598 877; 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., Bio/technology, 12:899-903,1988).

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., Immunol.Lett., 39:91-99,1994; U.S. Patent number 5,474,981; Gillies etc., PNAS, 89:1428-1432,1992 and Fell etc., J.Immunol., 146:2446-2452,1991, 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.

5.6 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 variant or its any derived protein.In another embodiment, 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.262:4429 4432).The method of introducing includes but not limited in intracutaneous, intramuscular, intraperitoneal, intravenously, subcutaneous, the nose, epidural and oral route.Compound can pass through any administration easily, for example by transfusion or large bolus injection, absorb 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 local infusion, topical application (for example with postoperative wound dressings coupling) in the operation, 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 aforementioned position) 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 send in controlled release system and pass.In one embodiment, (referring to Langer, ibid can to use pump; Sefton, 1987, CRCCrit.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); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen and Ball (editor), Wiley, New York (1984); Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem.23:61 (1983); Also referring to Levy etc., 1985, Science228:190; 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 (Science 249:1527-1533 (1990)) 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 or 2473 nucleotide sequence coded polypeptide, or Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964 and 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.

5.7 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 blood, serum, blood plasma, saliva, urine 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 ID NO: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:1,11,13,15,2471 or 2473 nucleotide sequences or its 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 hSARSmRNA 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 the hSARS of preservation searching number CCTCC-V200303 virus or have the hSARS virus of SEQ ID NO:15 genomic nucleic acid sequence), or makes up based on SEQ ID NO:1,11,13,2471 or 2473 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 and 6.8 joints in addition vide infra) subsequently.In preferred embodiments, primer comprises SEQ ID NO:2475 and 2476 nucleotide sequences or SEQ IDNO:2480 and 2481 nucleotide sequences.In preferred embodiments, thermal cycling be 94 ℃ 10 minutes, 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ circulations of 30 seconds are 40 times then, 72 ℃ 10 minutes.In preferred embodiments, primer comprises SEQ ID NO:2477 and 2478 nucleotide sequences.In preferred specific embodiments, the invention provides real-time quantitative PCR and test existing: from the total RNA of sample extraction with hSARS virus in the following detection of biological sample, carry out reverse transcription acquisition cDNA to extracting total RNA, use primer and fluorescence dye such as the SYBR of specific primer as having SEQ IDNO:3 and 4 nucleotide sequences ^_Green I (it can send fluorescence when non-specific when combining with double-stranded DNA) makes cDNA experience PCR reaction.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 joints vide infra).

The reagent of preferred detection hSARS 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 for 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 epi-position 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 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 (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 or polypeptide or coded polypeptide or geneome RNA in the test sample, and hSARS in the existence of the mRNA of hSARS in the control sample or polypeptide or coded polypeptide or geneome RNA and the testing sample or the mRNA of polypeptide or coded polypeptide or the existence of geneome RNA compared.

The present invention also comprises the test kit of the existence that detects hSARS in the testing sample or polypeptide of the present invention or nucleic acid.Described test kit can for example comprise the tagged compound or the reagent that can detect hSARS 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 epi-position; Optional (2) different second antibody, it is in conjunction with polypeptide or first antibody, and is conjugated on the detectable reagent.

For test kit based on oligonucleotide, it for example can comprise: (1) oligonucleotide, for example can detect the oligonucleotide of ground mark, 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 a kind of 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.

5.8 identify the shaker test of antiviral agent

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 at least at least at least at least at least at least at least 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, the compound of test inhibition or reduction hSARS viral growth suppresses or reduces the ability of other viral growth speed, to test 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 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 (beta-lactam and macrolide medicine, fluoroquinolone or tsiklomitsin) that covers typical case and atypical pneumonia.

Collect nasopharynx aspirate and serum sample from all patients.Can obtain paired acute phase and convalescent serum and ight soil on one's body from some patients.Lung's living tissue from a patient is handled, used for virus culture, RT-PCR, conventional organization pathological examination and submicroscopy.Nasopharynx aspirate, ight soil and the serum used for the microbiological test of 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 transhipment 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, observation of cell pathology effect in back 1 day of 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 virosome, but in not by the control cells of virus infection, do not have.From the isolating virosome 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 virosome.

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

6.5 antibody response to isolated viral

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 of acetone fixed 37 ℃ of following incubations 45 minutes.Use the salt water washing incubation cell of phosphoric acid buffer 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

Name	Date	Experiment numbers	Anti-SARS
				Patient A patient B patient C patient D patient E contrast F patient G	On March 7, on March 3,03 year on March 1,03 on February 13,03 year on March 18,03 on March 4,03 year on March 11,03 on March 6,03 year on March 14,03 on March 4,03 year on March 3,03 on March 6,03 on February 26,03 year in 03 on February 25,03	S2728 S2728 S2441 S2441 S3279 S3279 M41045 MB943703 M38953 KWH03/3601 M27124 MB942968 M38685 KWH03/2900	＜50 1,600 50 200 200 1600＜50 800＜50 800＜50＜50＜50 is suspicious

The blind test sample:
			1a * 1b 2a * 2b 3a * 3b 4a * 4b 5a * 5b	Acute stage convalescence acute stage convalescence acute stage convalescence acute stage convalescence acute stage convalescence	＜50 1600 50 ＞1600 50 ＞1600 ＜50 ＜50 ＜50 ＜50

6a * 6b

Acute phase convalescence

＜50 ＜50

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 Superscript II reversed transcriptive enzyme (Invitrogen) by being recommended in of manufacturer contain the 10pg degenerated primer (5 '-GCCGGAGCTCTGCAGAATTCNNNNNNN-3 ', N=A, T, G or C; SEQ ID NO:5) in the 20 μ l reaction mixtures 100ng purifying RNA is carried out reverse transcription.By the operation instruction purifying reverse transcription product of QIAquick PCR purification kit, be eluted among the 10mM Tris-HCl (pH 8.0) of 30 μ l then by the manufacturer.3 μ l purifying cDNA products are joined in the 25 μ l reaction mixtures that contain following composition: 2.5 μ l, 10 * PCR damping fluid, 4 μ l 25mM MgCl ₂, 0.5 μ l 10mM dNTP, 0.25 μ l AmpliTaqGold ^_Archaeal dna polymerase (Applied Biosystems), 2.5 μ Ci[α- ³²P] CTP (Amersham), 2 μ l, 10 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 l PCR 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, 1 * 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 l, 10 * PCR damping fluid, 4 μ l 25mM MgCl ₂, 0.5 μ l 10mM dNTP, 0.25 μ l AmpliTaqGold ^_Archaeal dna polymerase (Applied Biosystems), 1 μ l, 10 μ M primers (5 '-GCCGGAGCTCTGCAGAATT-C-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.With TOPO TA clone test kit (Invitrogen) clone PCR products, the plasmid that connects is transformed in TOP 10 intestinal bacteria (E.coli) competent cells (Invitrogen).PCR inserts fragment and checks order by manufacturer's (AppliedBiosystems) suggestion by BigDye cycle sequencing test kit, and the order-checking product is analyzed by automatic sequencer (AppliedBiosystems, 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, see Fig. 8 (SEQ ID NO:11 and 12) and Fig. 9 (SEQ ID NO:13 and 14) from the aminoacid sequence (being respectively SEQ ID NO:12 and 14) of hSARS virus acquisition other two kinds of partial sequences (SEQ ID NO:11 and 13) and deduction.

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 (SEQ ID NO:16,240 and 737) is seen Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107).The deduction aminoacid sequence of the SEQ ID NO:15 complementary sequence that obtains with whole three kinds of frames (SEQ NO:1108,1590 and 1965) is seen Figure 12 (SEQ ID NO:1109-1589,1591-1964 and 1966-2470).

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, Seto WH, Shortridge KF, Peiris JSM.Evaluation of Directigen Fly A+Btest for rapid diagnosis of influenza A and B virus infections.J ClinMicrobiol.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, BestebroerTM, Herfst S, Van Der Kemp L, Rimmelzwan GF, Osterhaus AD.Detection of influenza A virus from different species by PCRamplification of conserved sequences 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).

The 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:

1. RNA extracts

Extract test kit by the QIAquick viral RNA and from 140 μ l NPA samples, extract RNA, wash-out in 50 μ l elution buffers.

2. Reverse transcription

RNA 11.5μl

0.1M?DTT 2μl

5 * 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℃

3. PCR

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

cDNA 2μl

10mM?dNTP 0.5μl

10 * damping fluid, 5 μ l

25mM?MgCl ₂ 5μl

25 μ M forward primers, 0.5 μ l

25 μ M reverse primers, 0.5 μ l

AmpliTaq Gold ^_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.

4. 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 size: 182bp

The real-time quantitative PCR test

Pass through QIAamp ^_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 Superscript of 10 μ l wash-outs ^_II reversed transcriptive enzyme (Invitrogen) carries out reverse transcription by explanation.Complementary DNA is at SYBR then ^_Increase in Green I fluorescent reaction (Roche) mixture.Briefly, the cDNA, the 3.5mmol/L MgCl that contain 2 μ l 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 reactions 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).

6.8 the detection of hSARS virus N-gene among the patient

6.8.1 the RT-PCR diagnostic method of coronavirus among the SARS patient

Required equipment (96 samples):

The total RNA separation system of 1 * SV

2 * Mega titer plate

3 * 96-hole PCR plate

1 * 0.5-10 μ l multichannel pipettor

1 * 10-100 μ l multichannel pipettor

1 * 20-200 μ l multichannel pipettor

1 * vacuum pump

1 * have a swinging bucket rotor of trace test plate well-bucket

2 * PCR instrument (compatible) with the 96-orifice plate

1 * gel-electrophoretic apparatus

Stage 1 ^*-clinical sample is handled (1 medical personnel/clinical technician)

● 500 μ l virus transportation substratum (contains the 2g sodium bicarbonate in every liter of Earle balanced salt solution, 5g ox blood albumin, 200 μ g vancomycins, 18 μ g amikacins and 160U nystatin) in the sample equal portions from each object pack in the hole of 96-hole mega titer plate, 500 μ l lysis buffers (1 *) are arranged in the hole, wherein contain PK-15 cell (the ATCC CCL-33 in the necessary substratum of 100 μ l complete limits; 5.0 * 10 ⁵Cell/ml), (EMEM is Invirtogen) as internal contrast with Earle salt ^*

● by aspirating the mixed pyrolysis thing 3 times.

● enter the stage 2.

^* Stage 1 should carry out in III level Biohazard Safety Equipment.

^*At least should comprise in the platform of 96-hole that two negative samples are as negative control.

The total RNA of stage 2-extracts (1 Laboratory Technician)

● vacuum manifold is set.Board is placed on the vacuum manifold pedestal (Manifold Base).

● lysate is transferred to each hole of SV 96 boards (board) from the mega titer plate.

● apply vacuum and pass through board up to lysate.Remove vacuum.

● in each hole of board, add 500 μ l SV RNA washing solns (washing soln).

● apply vacuum and pass through board up to washing soln.Remove vacuum.

● be prepared as follows the DNA enzyme mixtures incubated that is used for whole 96-orifice plate:

Yellow kernel damping fluid (yellow core buffer) 2ml

0.09M?MnCl ₂ 250μl

DNA enzyme I 250 μ l

● the DNA enzyme mixtures incubated of 25 μ l prepared fresh directly is applied to the film of board.

● hatched 10 minutes at 20-25 ℃.

● in each hole of board, add 200 μ l SV DNA enzymes and stop solution.

● apply vacuum and stop solution up to SV DNA enzyme and pass through board.Remove vacuum.

● in each hole of board, add 500 μ l washing solns.

● apply vacuum and pass through board up to washing soln.Close vacuum.

● the centrifugal board of 3000 * g 30 seconds is to remove remaining washing soln.

● board is transferred to the top of 96-hole RT plate.

● in each hole of board, add 50 μ l nuclease free water with eluted rna.

● at room temperature hatched 1 minute.

● the centrifugal board of 3000 * g is 1 minute under 4 ℃.

● collect in RNA to the 96-hole RT plate of wash-out.

● in each hole of plate, add 5 μ l 3M sodium acetates and 200 μ l, 95% ethanol.

● the RT plate is placed on ice, hatched 15 minutes.

● the centrifugal plate of 3000 * g is 15 minutes under 4 ℃.

● by being inverted the plate supernatant discarded, on clean paper handkerchief, blot.

● precipitate with 200 μ l, 70% washing with alcohol.

● the centrifugal plate of 3000 * g is 10 minutes under 4 ℃.

● by being inverted the plate supernatant discarded, on clean paper handkerchief, blot.

● dry air precipitation 5 minutes.

● in each hole, add the water of 12 μ l nuclease free.

● of short duration vortex vibration plate, with dissolution precipitation (sample result is referring to Figure 18).

● enter the stage 3.

Stage 3-reverse transcription (1 Laboratory Technician)

● in the 1.5-ml test tube, be prepared as follows the general mixture of the RT that is used for whole 96-orifice plate (100 reactions):

	Each reaction	×100
			Sexamer at random, 3 μ g/ μ l DNTP, the 10mM first chain damping fluid, 5 *	0.05μl 1μl 4μl	5μl 100μl 400μl

DTT，0.1M Superscript?II，200U/μl	?2μl ?1μl	200μl 100μl
			Amount to	?8.05μl	805μl

● in 8 holes of the general mixture sheet in 96-hole that 100 μ l RT mixture five equilibriums are extremely clean.

● shift 8.05 μ l RT mixtures to each hole of RT plate that contains 12 μ l RNA from this plate, by mixing for 3 times with the multichannel pipettor suction.Change suction nozzle in each back of shifting.

● hatched sample 50 minutes for 42 ℃, hatched 15 minutes for 70 ℃ then.

● enter the stage 4.

Stage 4-N-gene specific PCR (1 Laboratory Technician)

● in two 2059 culture tubes, be prepared as follows the general mixture of the PCR that is used for whole 96-orifice plate (100 reactions):

	The N-specific PCR		Contrast PCR
					Each 25 μ l reaction	×100	Each 25 μ l reaction	×100
	mQH 2O 10 * PCR damping fluid 25mM MgCl 210mM dNTP forward primer 10 μ M reverse primers 10 μ M AmpliTaq Gold _The 500U template DNA	?18.65μl ?2.5μl ?1.5μl ?0.25μl ?0.5μl ?0.5μl ?0.1μl ?1μl	1865μl 250μl 150μl 25μl 50μl 50μl 10μl -	?17.65μl ?2.5μl ?2.5μl ?0.25μl ?0.5μl ?0.5μl ?0.1μl ?1μl					1765μl 250μl 250μl 25μl 50μl 50μl 10μl -
Amount to					?25μl	2400μl	?25μl	2400μl

● N-gene specific PCR and contrast PCR carry out in two independent PCR plates.

● with the general mixture five equilibrium of 290 μ l PCR to 96-hole PCR plate first row in.

● be listed as the general mixture five equilibrium of 24 μ l to each hole of PCR plate from first.

● 1 μ l cDNA template (from the stage 4) is transferred in each hole of PCR plate.

● by mixing for 3 times with the multichannel pipettor suction.Replace suction nozzle in each back of shifting.

● plate is sealed with sealant tape.

● in two 96-hole PCR instrument, carry out following reaction:

N-gene specific PCR Contrast PCR

94 ℃ 10 minutes 94 ℃ 10 minutes

72 ℃ 10 minutes 72 ℃ 10 minutes

● stage 5-gel electrophoresis (1 Laboratory Technician)

● 5 μ l N-gene specific PCR products and 5 μ l contrast PCR product are mixed with 1 μ l tetrabromophenol sulfonphthalein application of sample dyestuff.

● in the hole with sample pipetting volume to 2% sepharose.

● at 140V, 250mA makes PCR product electrophoresis 30 minutes.

● use the ethidium bromide staining gel.

● with UV colour developing product and write down the result.

6.8.2 use the primer of SEQ ID NO:2480 and 2481

Described in the 6.8.1 joint, carry out the RT-PCR diagnostic method, but carry out some modification.

From the clinical sample isolation of RNA

The clinical sample that comprises nasopharynx aspirate (NPA) and excrement sample is provided by Hong Kong University's department of microbiology.In addition, also collect to disperse thing and lung biopsy from the tracheae of three time points of crucial patient A described in the New Engl.J Med.348:1967-76 (Drosten C.S. etc., 2003).In civilian hospital, carried out sample collection to April 28 from April 1st, 2003.The method of sample collection is as (also referring to Poon etc., 2003, ClinicalChemistry 49:953-955) as described in the previous section.From clinical sample extract total RNA with the total RNAIsolation System of SV96 to the scheme of producer carry out following modification carry out (Promega, WI, USA).500 μ l virus transportation substratum (contains the 2g sodium bicarbonate in every liter of Earle balanced salt solution, 5g ox blood albumin, 200 μ g vancomycins, 18 μ g amikacins and 160U nystatin) in NPA/ excrement sample mix with equal-volume SV RNA lysis buffer, contain pig kidney epithelium (PK-15) cell (the ATCC CCL-33 of 100 μ l complete limits in must substratum in this damping fluid; 5.0 * 105 cells/ml), (EMEM is Invitrogen) as internal contrast with Earle salt.Mixture is transferred in the hole of SV 96 boards.With after the 500 μ l SVRNA washing solns washings, before the elution step, plate centrifugal 30 seconds of 3000 * g to remove residual washing soln.RNA is with 50 μ l nuclease free water elutions then, and by with plate at 3000 * g centrifugal 1 minute, RNA is collected in the clean 96-hole PCR plate.By in the presence of 5 μ l 3M sodium acetates and 200 μ l, 95% ethanol, hatching 15 minutes, concentrate the RNA of wash-out on ice.4 ℃ 3000 * g is after centrifugal 15 minutes down, and RNA precipitates with 200 μ l75% washing with alcohol, is dissolved in the 12 μ l nuclease free water.The RNA that extracts reverse transcription immediately becomes first chain of cDNA.

The first chain cDNA's is synthetic

Use the Superscript of 200U ^_(Invitrogen USA), carries out reverse transcription in the 20 μ l reaction solutions of sexamer, RT damping fluid (1 *), 10mM dithiothreitol (DTT) (DTT) and 0.5mM triphosphate deoxy-nucleotide (dNTP) containing 0.15 μ g to the II reversed transcriptive enzyme at random.Be reflected among the Peltier Thermal Cycler (MJ Research) and carry out with following condition: 42 ℃ 50 minutes, then 70 ℃ 15 minutes.

Polymerase chain reaction (PCR)

Full SARSCoV genome sequence design primer according to the local sample HK-39 (searching number AY278491) of former declaration.Forward primer (SRS251:5 '-GCAGTCAAGCCTCTTCTCG-3 '; SEQ ID NO:2480 is the genomic 28658-28676 Nucleotide of CCTCC200303 corresponding to HK-39SARS), reverse primer (SRS252:5 '-GCCTCAGCAGCAGATTTC-3 ', SEQ ID NO:2481; Corresponding to the genomic 28866-28883 Nucleotide of HK-39 SARS) amplify the 225bp fragment from the N-gene regions, this fragment and other coronavirus do not show homology.The primer of cloning RNA RNA-dependent polysaccharase (1b gene) as parallel control (coro3:5 '-TACACACCTCAGCGTTG-3 ' (SEQ ID NO:3), corresponding to 18041-18057 Nucleotide; And coro4:5 '-CACGAACGTGACGAAT-3 ' (SEQ ID NO:4), corresponding to 18207-18222 Nucleotide, Hong Kong University's department of microbiology).Two amplicons all are cloned into identical pCR2.1 cloning vector (Figure 17).The plasmid of serial dilution is used to measure dynamicrange and the top condition (Figure 21 A and 21B) of PCR.Another set of from internal contrast (actin-F:5 ' TGAGACCTTCAACACGCC-3 ' (the SEQ ID NO:2482) of the segmental primer of pig beta-actin gene amplification 745bp as diagnosis PCR test; And actin-R:5 '-ATCTGCTGGAAGGTGGAC-3 ' (SEQ ID NO:2483)).

Conventional PCR and gel electrophoresis are carried out as preliminary experiment.Speak briefly, use 0.5U reorganization Taq archaeal dna polymerase (Invitrogen Life Technologies), containing PCR damping fluid (1 *), 1.5mM MgCl ₂, 0.1mM dNTPs and forward and each 0.5pmol of reverse primer 25 μ l reaction solutions in amplification from 1 μ l cDNA of clinical sample.Be reflected among the PeltierThermal Cycler (MJ Research) and carry out with following condition: 94 ℃ 3 minutes, then 94 ℃ 10 seconds, 56 ℃ 10 seconds, 72 ℃ 10 seconds, 50 circulations, and 72 ℃ of final prolongation steps of following 10 minutes.Amplicon is with 2% agarose gel electrophoresis analysis (Figure 23).Quantitative PCR in real time SYBR in the diagnosis of clinical sample ^_Green fluorescence group carries out.In 25 μ l reaction solutions, 1 μ l cDNA template and the general mixtures of 12.5 μ l (2 *) Green PCR (Applied Biosystems) and forward and each 0.5pmol of reverse primer mix.The reaction solution volume transfers to 25 μ l with distilled water.Be reflected in the iCycler iQ PCR in real time detection system (Bio-Rad) and under the condition identical, carry out with conventional PCR.In the PCR working cycle, (FAM excites=490nm, emission=530nm) (Figure 22 A) to collect fluorescent signal when each prolongs the step end.Use the maximum curvature method to measure the threshold cycle number (Ct) of each sample.Carry out curve analysis (Figure 22 B) after 10 minutes the final prolongation.Non-SARS patient comprises the patient who infects adenovirus (n=5), respiratory syncytial virus (n=5), human stroma lung virus (n=5), influenza A virus (n=5) or influenza B virus (n=5), from their cDNA as the negative control of test.

Rna blot analysis

The Vero cell that SARS-CoV HK-39 strain is infected is provided by Hong Kong University's department of microbiology.Use TRIzol ^_Reagent (Invitrogen Life Technologies) according to the method for producer from the total RNA of cell extraction.By containing the total RNA of electrophoretic separation 8 μ g on 1% sepharose of 3.7% formaldehyde.RNA is transferred on the positive polarity nylon membrane (Roche DiagnosticCorporation) by the kapillary trace, and is crosslinked fixing by UV.Be used as probe synthetic template with identical RNA sample synthetic cDNA.Four pairs from 1b (Nucleotide 18057-18222; SEQ ID NO:2484), S (Nucleotide 21920-22107; SEQ ID NO:2485), M (Nucleotide 26867-26996; SEQ ID NO:2486) and N (Nucleotide 28658-28883; SEQ IDNO:2487) it is synthetic that the segmental primer of gene amplification is used for probe.The detection of DIG-mark, hybridization and the band of probe is carried out according to the method for producer with digoxin system (Roche Molecular Biochemcials).Signal is with chemoluminescence method analysis (Figure 24).

Result and discussion

Extensive RT-PCR test provides the fast method of monitoring and examination SARS suspected patient.This result can be used for replenishing the clinical diagnosis assessment.For reaching diagnostic purpose, test should be reliably, and its accuracy should obtain guaranteeing, to prevent the appearance of false negative and false positive results.But accuracy of test can be subjected to the influence of some factor.The common technical problem of PCR is the amplification failure (referring to Figure 21) that the existence owing to the PCR inhibitor causes.

These PCR inhibitor comprise the protoheme compound of finding in the blood, watery and vitreous humour, heparin, EDTA, urine and polyamines (Fredricks etc., 1998, J Clin.Micro.36:2810-16).Current, NPA or excrement sample collection are in the transportation substratum, to keep the activity of virosome.In preliminary experiment, when the total RNA that extracts is directly used in the first chain cDNA when synthetic without any processing, RT-PCR is suppressed (27 sample in 25).But after the simple ethanol sedimentation step, the amplification of DNA can obtain keeping (Figure 19).Use SV or the total RNA separation system of SV96 can obtain identical result (data not shown).This shows that some component in substratum or the NPA/ excrement sample can influence the downstream process of diagnostic test.

In addition, this sample collection method has been diluted the virus titer in the sample, especially infecting in early days, (Drosten etc. when the virus titer in the N﹠T wipe samples is low, 2003, New England Journal of Medicine, on the internet at http://content.nejm.org/cgi/reprint/NEJMoa030747v2).The susceptibility that this hint is used for the PCR test of SARS relies on the quality of sample and the time that lysis is tested.For increasing the susceptibility of test, before the first chain cDNA is synthetic, concentrate from the isolating total RNA of clinical sample.

For fear of RNA separate and the first chain cDNA building-up process in the false negative PCR result that causes of failure, parallelly extract total RNA from clinical sample and PK-15 mammalian cell.Figure 23 represents 48 clinical samples are comprised the RT-PCR The selection result of NPA and excrement sample.PCR is parallel with beta-actin PCR carries out in diagnosis.All samples is positive in beta-actin PCR.This result shows, can be successfully from sample extraction and synthetic RNA and cDNA in one-step method disclosed herein.According to this internal contrast, guaranteed that this has eliminated the false negative that is caused by the failure of any in the said process from total RNA of sample separation and synthetic cDNA.And, the 96-hole test form of this paper exploitation can adopt high-throughput screening method, we can be obtained to surpass the diagnostic result of 90 clinical samples by 1 clinical staff in 3 hours like this, and existing method is at the independent sample of processing in vitro, and each technician can only handle about 30-50 sample every day.

Therefore the real-time quantitative PCR test is used to the SARS-CoV diagnostic purpose than traditional agarose gel electrophoresis associating PCR test more responsive (Poon etc., J Clin.Virol.28:233-8).Among N-gene and the 1b-gene specific PCR, detected positive signal in 38 of 136 clinical samples of selecting at random.In these 38 positives, 3 is excrement sample (2.2%), and 35 is NPA sample (25.7%).Use the verification and measurement ratio of test of N-gene specific RT-PCR as shown in table 2 in different time points.

Table 2

Start Date	Sample number	Number positive	Verification and measurement ratio (%)
				1-2 3-4 5-6 7-8 9-10 negative control	15 17 15 1 9 19	24454 total negatives	13.3 23.5 26.7 38.5 44.4 --

Confirm the certainty of these 38 positive cases by the curve analysis of PCR product.The specificity melting temperature(Tm) of N gene and 1b gene PCR product (being respectively 85.5 ℃ and 80.5 ℃) shows, has amplified target fragment in reaction.The specificity of test also is confirmed by non-SARS patient's sample, and described patient comprises the patient of suffer from adenovirus (n=5), respiratory syncytial virus (n=5), human stroma lung virus (n=5), influenza A virus (n=5) and influenza B virus (n=5).Does this result show that all these samples are negative (figure in mensuration?).These results show that N-gene specific RT-PCR test is specific to the diagnosis of SARS-CoV.

In addition, we prove that also N-gene specific PCR is more responsive than pcr amplification 1b rna polymerase gene.At first with the amplification condition (referring to Figure 22) (referring to Figure 20) of two kinds of PCR tests of the plasmid construction thing optimization that contains 1: 1 1b-and N-gene fragment.Do you obtain the dynamicrange (figure of N-gene specific PCR?), find that its Ct value is lower than the Ct value of 1b-specific PCR.This shows that N-gene specific PCR can reach the higher amplification efficiency than 1b-gene specific PCR when using the template of identical copies number.Use cDNA to carry out PCR then from the Vero cell of clinical sample or virus infection.Figure 22 A represents to disperse thing and the N gene of lung biopsy generation and the Ct and the half maximum value of 1b gene specific PCR fluorescent signal by NPA, tracheae from patient A.This result shows, in all positive, the fluorescent signal that produces among the N gene specific PCR is than high in the 1b specific PCR (average 26.0%, change in 6.3-60%).In addition, in most of SARS-CoV positive, the Ct value of N gene specific PCR is than 1b specific PCR low (0.1-4.6 circulation) (table 3).

Table 3

S/N	N	1b	ΔCt	S/N	N	1b	ΔCt	S/N	N	1b	ΔCt	S/N	N	1b	ΔCt
																56851 55751 62290 55531 55963 65733 34862 32814 33935 34861	27.1 27.6 41.9 41.0 41.7 43.6 33.5 38.6 35.3 31.4	27.8 27.7 43.2 43.1 42.3 44.9 33.7 40.8 36.5 31.7	0.6 0.1 1.3 2.1 0.6 1.3 0.2 2.2 1.2 0.3	34862 45971 45972 45973 69145 56386 55527 56851 69073 67423	31.9 43.6 43.6 42.1 27.2 32.8 37.3 23.9 24.3 28.7	33.2 48.2 46.5 43.2 27.7 33.8 39.4 26.1 26.1 29.4	1.3 4.6 2.9 1.1 0.5 1.0 2.1 2.2 1.3 0.7	67429 67438 68116 68118 68134 68184 68185 68187 68788 68791	35.3 28.4 30.0 36.7 32.4 30.6 27.5 40.3 35.5 34.8	35.6 27.5 33.5 37.7 33.2 32.4 30.1 41.5 37.6 35.3	0.3 -0.9 3.5 1.0 0.8 1.8 2.6 1.2 2.1 0.5	68796 68798 68800 68801 70562 70589 70591 70059 ? ?	32.4 28.8 34.6 31.9 40.2 35.5 36.0 41.4 ? ?	34.5 28.4 38.3 32.8 43.3 38.2 38.2 43.1 ? ?	2.1 -0.4 3.7 0.9 3.1 2.7 2.2 1.7 ? ?

Δ Ct=1.49 ± 0.47,95% fiducial interval=0.74-2.23 (F-check)

Statistical analysis shows that the Ct of N-gene PCR test significantly is lower than ab-gene test (95% fiducial interval=0.74-2.23, F-check).Fluorescent signal that N gene specific PCR is stronger and lower Ct value provide more sensitive diagnostic result and a large amount of target for test.

Use is from the cDNA of the Vero cell of SARS-CoV infection, and the amplification curve shown in Figure 21 B has shown the difference between N gene and the 1b gene specific PCR.The Ct of N gene and 1b gene specific PCR is respectively 35.3 and 37.8.This phenomenon has two major causes: (1) N expression of gene level is than 1b gene height; (2) copy number of N gene is more much higher than 1b gene, because the copy of a N gene is arranged behind each transcript in the SARS-CoV cells infected.Rna blot analysis has been supported this hypothesis (Figure 24).When N-gene specific PCR product when the probe, at least 5 kinds of transcripts from virus are hybridized and are obtained positive signal (Figure 24).This result is consistent with following discovery, uses the probe derive from 3 ' untranslated district to detect 5 kinds of subgenomic mRNAs (Rota etc., 2003, Science 300:1394-99) by the Northern hybridization from the RNA of SARS-CoV cells infected in this discovery.On the other hand, when 1b PCR product is used as probe, have only 2 kinds of macromolecule transcripts to be hybridized, show that the copy number of N gene when transcribing with genetic expression is more much higher than 1b gene in host cell.The Northern results of hybridization is supported strongly to draw a conclusion: as the target of diagnosis screening, the pcr amplification district is more responsive than other zone in the N gene of SARS-CoV.The amplification that might surpass a kind of genome area can increase the specificity of detection (YamW.C. etc., 2003, J:Clin.Microbiol.41:4521-24).

In a word, we have developed RT-PCR diagnosis detecting method of new generation, and it is sensitiveer than traditional diagnostic test for detecting the relevant coronavirus of SARS.Test method of the present invention provides high-throughput, hypersensitivity screening platform, make our scale be extended to can be in single operating line every day detect thousands of doubtful SARS cases.Add in test the PK-15 cell and can strengthen the susceptibility and the accuracy of test as internal contrast and N gene and 1b gene as the use of diagnosis target.We have adopted the method for 96-hole real-time quantitative PCR and order-checking form, test required time to shorten, and obtain the information of viral genotypic change.

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 median ages 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 4) 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.During initial the inspection, auscultation 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 4

Clinical symptom	Patient's number (per-cent)
		Feel cold or the cough myalgia uncomfortable rhinorrhea of the shivering with cold anorexia diarrhoea headache and dizzy of breathing hard of having a sore throat	50(100％) 37(74％) 31(62％) 27(54％) 25(50％) 12(24％) 10(20％) 10(20％) 10(20％) 5(10％) 10(20％) 6(12％)

^*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 5).Liver parenchyma enzyme alanine aminotransferase (ALT) and muscle enzyme creatinine kinases (CPK) raise in 34% and 26% case respectively.

Table 5

The chemical examination parameter	Mean value (scope)	Unusual per-cent	Normal range
				Hemoglobin anaemia white blood cell count(WBC) leucocyte reduces the remarkable lymphocyte of LC and reduces (＜1.0 * 109/ L) the low albumin globulin globulin rising kreatinin kinases kreatinin kinases of platelet count decrease of platelet alanine aminotransferase (ALT) ALT raising albumin raises	12.9(8.9-15.9) ? 5.17(1.1-11.4) ? 0.78(0.3-1.5) ? ? 174(88-351) ? 63(11-350) ? 37(26-50) ? 33(21-42) ? 244(31-1379) ?	? 9(18％) ? 13(26％) ? 34(68％) ? ? 20(40％) ? 17(34％) ? 34(68％) ? 10(20％) ? 13(26％)	11.5-16.5g/dl ? 4-11×10 9/L ? 1.5-4.0×10 9/L ? ? 150-400×10 9/L ? 6-53U/L ? 42-54g/dl ? 24-36g/dl ? 34-138U/L ?

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 6).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 to begin to take 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 6

	Concurrent case (n=19)	There is not concurrent case (n=31)	The P value
				On average family's contact symptom on average (SD) when being admitted to hospital that touches society is visited in (scope) the male/female ratio potential disease way of contact of (SD) age to China travelling medical and nursing work person to hospital	49.5±12.7 8/11 5 _? 1 5 1 8 4 5.2±2.0	39.0±10.7 14/17 1 _? 3 9 4 5 10 4.7±2.5	P＜0.01 N.S P＜0.05 ? N.S. N.S. N.S. P＜0.05 N.S. N.S.

Time length (my god) average (SD) be admitted to hospital temperature (℃) average (SD) initial total periphery WBC counting (* 10 9/ L) average (SD) initial lymphocyte count (* 10 9/ L) thrombocytopenicly have (＜150 * 10 9/ L) the impaired inspection of liver function CXR changes (quantity of involved area) and average (SD) fate § that the state of an illness worsens occurs from symptom and Ribavirin and steroids occur to average (SD) fate that begins to give Ribavirin and steroids beginning to give after sb.'s illness took a turn for the worse to Ribavirin and steroids respond result's improvement or the ‖ that do not take a turn for the better that fully recovers from symptom

? 38.8±0.9 5.1±2.4 ? 0.66±0.3 ? 8 ? 11 1.4 ? 8.3±2.6 ? 7.7±2.9 ? ? 12 ? 11 ? ? 10 8

38.7 ± 0.8 5.2 ± 1.8 0.85 ± 0.3 12 6 1.2 inapplicable 5.7 ± 2.6 0 28 31 0

? N.S. N.S. ? P＜0.05 ? N.S. ? P＜0.01 N.S. ? ? ? ? P＜0.05 ? ? P＜0.001 ? P＜0.05 ? ? P＜0.01 P＜0.01

^*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 0.7 * 10 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 (2.7 * 10 ⁹/ L), lymphopenia (0.6 * 10 ⁹/ L) and thrombopenia (104 * 10 ⁹/ L).Cover although carry out empirical antimicrobial drug 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 body, 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 virosome (Fig. 5 B) of 55-90nm that diameter is arranged in the slide wall vesicle in the tenuigenin.In the also visible virosome 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 virosome in the tenuigenin of its furfur cell.These virosome the size with morphology on to observed virosome 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 pairs 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, makes it carry 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 results 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 6.48 * 10 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.The false-positive case that comprises beginning, the sensitivity of real-time quantitative PCR test is 79%, 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 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 OC43 in 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, these primers there is no fear of and OC43 strain generation cross reaction very much.

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.

When detecting the evidence that viral RNA all is considered to infect hSARS to the HP-CV seropositivity or in NPA or ight soil in the single serum, there are 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.

Discuss

Breaking out of SARS is uncommon in many aspects, especially occurs pneumonia patient colony 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 (SEQ ID 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.Eds Knipe DM of this virus then more for certain, Howley PM Fields Virology, the 4th edition, Lippincott Williams﹠amp; Wilkins, Philadelphia, 1187-1203).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.Whether there are other microbiological factor or other cofactor in this advancing of disease, to have an effect and are 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 Lippincott Williams﹠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 in hospitaliziedpatients with " common cold " virus infections.Clin Infect Dis.2000; 31:96-100; With Foltz EJ, Elkordy MA.Coronavirus pneumonia followingautologous 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 OC43 and 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, Arbour N.Humancoronavirus OC43 and 229E persistence in neural cell cultures and humanbrains.Adv Exp Med Biol. waits to deliver).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 ight soil, 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, ChanPKS, Peiris JSM etc., Clinical features and rapid viraldiagnosis of humandisease associated 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 symptom 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).

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 is attached in this paper specification sheets by reference, and its degree clearly and is individually pointed out to be 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 (29)

1. isolated nucleic acid molecule, described nucleic acid molecule is made up of SEQ ID NO:2471 nucleotide sequence or its complementary sequence substantially.

2. isolated nucleic acid molecule, described nucleic acid molecule is made up of SEQ ID NO:2473 nucleotide sequence or its complementary sequence substantially.

3. isolated nucleic acid molecule, described nucleic acid molecule under stringent condition with have the nucleotide sequence of claim 1 or 2 or the making nucleic acid molecular hybridization of its complementary sequence.

4. claim 1,2 or 3 each nucleic acid molecule, wherein said molecule is RNA.

5. claim 1,2 or 3 each nucleic acid molecule, wherein said molecule is DNA.

6. isolated nucleic acid molecule, described nucleic acid molecule under stringent condition with nucleic acid molecule or its complementary sequence hybridization of claim 1 or 2, the aminoacid sequence of wherein said nucleic acid molecule encoding biologically active, described biological activity is by SEQ ID NO:2471 or 2473 nucleotide sequence coded polypeptide performances.

7. isolated polypeptide, described polypeptide is by the nucleic acid molecule encoding of claim 1 or 2.

8. an antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the N-gene protein of hSARS virus.

9. an antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the S-gene protein of hSARS virus.

10. claim 8 or 9 each antibody or its Fabs are in described antibody or its Fab and hSARS virus.

11. an antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of claim 7.

12. one kind is detected the method that hSARS virus N-gene exists in biological sample, described method comprises:

(a) compound of the described N-gene of sample and selective binding is contacted;

(b) whether detect described compound in conjunction with the gene of N-described in the sample.

13. the method for claim 12, wherein the compound in conjunction with described N-gene is the nucleic acid molecule that comprises nucleotide sequence or its complementary sequence, and described nucleotide sequence has at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150 or 1200 continuous nucleotides of SEQ IDNO:2471 nucleotide sequence.

14. the method for claim 12, wherein the compound in conjunction with described N-gene is the nucleic acid molecule that comprises SEQ ID NO:2475,2476,2480 and/or 2481 nucleotide sequences.

15. a method that detects the existence of hSARS virus S-gene in biological sample, described method comprises:

(a) compound of the described S-gene of sample and selective binding is contacted;

(b) whether detect described compound in conjunction with the gene of S-described in the sample.

16. the method for claim 15, wherein the compound in conjunction with described S-gene is the nucleic acid molecule that comprises nucleotide sequence or its complementary sequence, and described nucleotide sequence has at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200,2000 or 3000 continuous nucleotides of SEQ IDNO:2473 nucleotide sequence.

17. the method for claim 15, wherein the compound in conjunction with described S-gene is the nucleic acid molecule that comprises SEQ ID NO:2477 and/or 2478 nucleotide sequences.

18. the method that the polypeptide of a test right requirement 7 in sample exists, described method comprises:

(a) sample is contacted with the compound of the described polypeptide of selective binding;

(b) whether detect described compound in conjunction with polypeptide described in the sample.

19. the method for claim 18, wherein the compound in conjunction with polypeptide is an antibody.

20. a method of identifying hSARS virus infection object, described method comprises:

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

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

(c) overlap the primer that derives from hSARS N-gene nucleotide series with one cDNA is carried out the PCR in real time test.

21. the method for claim 20, wherein said primer have SEQ ID NO:2475 and 2476 nucleotide sequences respectively.

22. the method for claim 20, wherein said primer have SEQ ID NO:2480 and 2481 nucleotide sequences respectively.

23. a method of identifying hSARS virus infection object, described method comprises:

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

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

(c) overlap the primer that derives from hSARS S-gene nucleotide series with one cDNA is carried out the PCR in real time test.

24. the method for claim 23, wherein said primer have SEQ ID NO:2477 and 2478 nucleotide sequences respectively.

25. a test kit, described test kit comprise one or more isolated nucleic acid molecule in one or more containers, described nucleic acid molecule comprises SEQ ID NO:2475 and/or SEQ IDNO:2476 nucleotide sequence.

26. a test kit, described test kit comprise one or more isolated nucleic acid molecule in one or more containers, described nucleic acid molecule comprises SEQ ID NO:2480 and/or SEQ IDNO:2481 nucleotide sequence.

27. a test kit, described test kit comprise one or more isolated nucleic acid molecule in one or more containers, described nucleic acid molecule comprises SEQ ID NO:2477 and/or SEQ IDNO:2478 nucleotide sequence.

28. a test kit, described test kit comprise the antibody of one or more claims 8 or 9 in one or more containers.

29. a test kit, described test kit comprise the antibody of one or more claims 11 in one or more containers.

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