CN1572798A - SARS coronary virus related polypeptide and its uses - Google Patents
SARS coronary virus related polypeptide and its uses Download PDFInfo
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- CN1572798A CN1572798A CN 03129289 CN03129289A CN1572798A CN 1572798 A CN1572798 A CN 1572798A CN 03129289 CN03129289 CN 03129289 CN 03129289 A CN03129289 A CN 03129289A CN 1572798 A CN1572798 A CN 1572798A
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Abstract
The present invention relates to a SARS coronary virus related polypeptide and its uses. Concretely, the invention uses polypeptide from SARS virus N albumen synthesized by manpower as antigen and the antibody (single anti, multi-anti) generated after the polypeptide is used for immunity for animals, and combines enzyme linked immunity and other methods to detect the early anti-SARS antibody IgM or IgG in human blood serum sample, or directly detect the SARS pathogen in the blood serum sample.
Description
Technical field
The present invention relates to sars coronavirus related polypeptide and application thereof.Particularly, the present invention uses artificial synthetic to be derived from the polypeptide of SARS virus nucleocapsid protein (N albumen), and the antibody that produces behind this polypeptide immune animal (monoclonal antibody, how anti-), in conjunction with early stage anti-SARS antibody IgM or the IgG among enzyme linked immunological and the additive method detection human serum sample, or directly detect SARS pathogenic agent in the serum sample.
Background technology
Atypical pneumonia is that severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, confirm recently, is a kind of coronavirus by pathogenic agent SARS).This virus is a kind of positive chain RNA virus, and genome is a single stranded RNA, contains Poly (A), and length overall is near 30kb.The virus surface club shaped structure about 20nm of having an appointment is the imperial crown shape.This pathogenic agent has very strong infectivity and infectivity, and virus through after the early stage reproduction process, causes extensive inflammatory reaction in lung in patient's body, finally causes lung tissue's damage.Owing to clinically lack effective medicine, be effectively preventing means so present stage isolates for treatment infected patient rapidly.
The diagnostic method that has developed at present and used comprises:
1.RT-PCR check virus.Can in time check the carrier, after recovery from illness, show negative.But be subjected to the restriction of plant and instrument, easily produce more false negative and false positive and cause failing to pinpoint a disease in diagnosis or mistaken diagnosis.
2. by the diagnosis of clinical symptom comprehensive evaluation.This method is early stage disease, and is not obvious because of symptom, and can't diagnose accurately.In the late period of disease,, incured loss through delay disease has effectively been treated though can accurately diagnose.
3. seroimmunity is checked antiviral antibody (fluorescent immune method or ELISA).Antibody produces need be after morbidity at least 14 days, and therefore, this method can not be done early diagnosis.But from the immunology angle, body produces the pathogen specific antibody IgM early, selects suitable antigen with it in conjunction with also being used as the diagnosis reference.
The great majority that using at present are first three methods, and these methods can not be judged and differentiation patient and suspected case in clinical use easy, fast and accurately.
4. Detection of antigen (ELISA).Antibody (monoclonal antibody is with how anti-) by the preparation pathogen specific detects pathogenic agent in patient blood or the tissue, and this method accuracy rate height is easy and simple to handle, is fit to clinical expansion.But the specific antibody that lacks pathogenic agent at present.
Therefore, this area presses for exploitation new more effectively method and the test kit of early diagnosis SARS.
Summary of the invention
The object of the invention just provides a kind of quick, easy, SARS detection method and test kit that accuracy rate is high.
In a first aspect of the present invention, a kind of polypeptide that is derived from SARS virus is provided, its aminoacid sequence is SEQID NO:1,2 or 3.These SARS specific polypeptides can be specifically and the antibodies of anti-SARS virus.
In a second aspect of the present invention, a peptide species conjugate is provided, described conjugate is made of polypeptide, linking agent and BSA, and wherein said amino acid sequence of polypeptide is SEQ ID NO:1,2 or 3, and described linking agent is selected from glutaraldehyde or EDAC.
In a third aspect of the present invention, a kind of antibody is provided, described antibodies specific ground combines with SARS specific polypeptide of the present invention.
In a preference, described antibody is monoclonal antibody.
In a fourth aspect of the present invention, a kind of detection kit is provided, it contains antibody or its combination of SARS specific polypeptide of the present invention, polypeptide conjugate, anti-polypeptide of the present invention.
In a fifth aspect of the present invention, the method that whether has SARS virus or anti-SARS virus antibody in a kind of vitro detection sample is provided, comprise step:
(a) sample is contacted with the material that is selected from down group: the antibody of SARS specific polypeptide of the present invention, polypeptide conjugate, anti-polypeptide of the present invention or its combination;
(b) detect whether form antigen-antibody complex, wherein form mixture and just represent to exist in the sample SARS virus or anti-SARS virus antibody.
In a preference, described sample is serum sample or sputum.
In another preference, described material is that aminoacid sequence is SEQ ID NO:1,2 or 3 polypeptide.
In another preference, described material is a monoclonal antibody.
In another preference, the detection method of step (b) is a fluorescence detection.
Description of drawings
Fig. 1 has shown after SARS virus infects, IgG and IgM production of antibodies situation.
Fig. 2 has shown the proteic structural analysis to SARS virus S.
Fig. 3 has shown the recombinant N protein of purifying and SDS-PAGE electrophoretogram (12%SDS-PAGE electrophoresis, the coomassie brilliant blue staining of GST reference protein.)。Wherein swimming lane 1: the low molecular weight protein (LMWP) standard substance; Swimming lane 2:GST reference protein; Swimming lane 3: the recombinant N protein behind the purifying.
Mr. Fig. 4 the peptide section induce the antibody of generation and sars coronavirus N protein affinity to detect (Western Blot experiment) result.Wherein, swimming lane 1,3,5: the recombinant N protein behind the purifying; Swimming lane 2,4, the 6:GST reference protein.Swimming lane 1,2 with peptide section N1 immunity after rabbit anteserum be one anti-; Swimming lane 3,4 with peptide section N2 immunity after rabbit anteserum be one anti-; Swimming lane 5,6 with immunity before rabbit anteserum be one anti-.Albumen behind the 12%SDS-PAGE electrophoresis electrotransfer to pvdf membrane.After the sealing, adding corresponding rabbit anti-serum (1/1000 dilution), is two anti-with the IgG of the goat-anti rabbit of HRP mark, the ECL colour developing.
Embodiment
The present invention is by the sequential analysis to the potential antigen protein of SARS virus, found and had the very aminoacid sequence of strong antigen, by synthetic these the potential antigen target position of synthetic polypeptide, the antibody that obtains by these polypeptide immunes again, develop a kind of passing through and detect SARS virus or anti-SARS virus antibody, carry out the enzyme-linked immunoassay method and the test kit of clinical early stage SARS diagnosis.The present invention can significantly improve diagnosis efficiency, and easy, accuracy rate is high.
Referring to Fig. 1.Existing studies show that: virus reaches about ten days in human body duplicates the peak, and SARS-IgM antibody occurs in the time of 10～14 days also peaking very soon in morbidity, have 1/3 patient still can detect IgM antibody in the time of 60 days approximately, this antibody disappears substantially among most of patient.IgG antibody also can detect in the time of 10-14 days, peaked in the time of 60 days, still maintained high level in the time of 90 days.Can after patient infection, make efficient diagnosis in the shortest time so adopt the antibody test pathogenic agent; Adopt IgM to detect and about a back week of infection, to make diagnosis; It is then least desirable to detect the interior IgG of patient body.
In order to obtain to detect required related antigen of SARS virus and antibody, the inventor is by to genomic analysis of SARS virus and comparison, in conjunction with documents and materials recently and in the past, the potential antigen protein of SARS analyzed.
By with analysis and the comparison of coronavirus, the inventor finds that coronavirus is merged with cytolemma immediately by combining with cell surface receptor, virus particle enters cell.The life cycle of virus carries out in tenuigenin fully, does not need nucleus to participate in.SARS virus coding two big proteinoid itself, structural protein and Nonstructural Protein:
(a) structural protein mainly comprise the S (the main composition composition of club shaped structure) that is combined in the viromembrane surface, M albumen, and the N albumen that combines formation spirrillum nucleocapsid spline structure with RNA.
(b) Nonstructural Protein then is that viral RNA duplicates closely-related polysaccharase.
Among numerous albumen of expressing viral, S, M albumen have constituted viral outermost structure, and S albumen is a kind of crucial structural protein, and numerous coronavirus is widely different in this part, and this may be relevant at different target cells with it.
Referring to Fig. 2.S albumen is divided into S1, and S2 two portions wherein show by sequential analysis, and the S1 district comprises: 1-668; S2 district: 669-1162.S1 albumen it mainly with receptor in target cell in conjunction with relevant; S2 albumen then mediates the fusion of virus and cell.Because S albumen for the importance of virus infection, therefore filters out the neutrality antibody that can be used in treatment probably therein; And known coronavirus neutrality antibody all is to act on the S protein region.
Therefore, the present invention selects the main research object of the Spike albumen (S albumen) of SARS as the antigen target position, consider M simultaneously, N albumen also has the potential antigenicity, appropriate selection incomplete antigen decision epi-position, from clinical angle, the antibody that these positions produce may some meaning for the early diagnosis of disease.
After determining target protein, the present invention uses multiple antigenicity analysis software and website to the albumen complete sequence, wetting ability (high-hydrophilic) and the multilevel hierarchy of taking all factors into consideration aminoacid sequence (mostly are alpha-helix, beta sheet), those are present in the film surface portion preferential investigation, rejecting is included in the part in the film, has selected part of polypeptide sequence (table 1) like this from the N protein sequence.Detect N albumen and just show that virus is in replicative phase.
As used herein, " polypeptide of the present invention " or " SARS specific polypeptide " refers to the polypeptide that aminoacid sequence is as shown in table 1.
Polypeptide of the present invention can be used the ordinary method synthetic, also available recombination method production.
Polypeptide of the present invention can be directly used in the antibody that detects anti-SARS, also can be used for the albumen coupling with the BSA equimolecular quantity, thereby forms the polypeptide conjugate.Usually, described conjugate is made of polypeptide, linking agent and BSA, the preferred glutaraldehyde of wherein said linking agent (when the N-with polypeptide of the present invention holds coupling), EDAC (when the C-with polypeptide of the present invention holds coupling).
Conjugate of the present invention can be used for animals such as immunize rabbit, mouse, thereby obtains the antibody (" antibody of the present invention ") of anti-polypeptide of the present invention.Antibody of the present invention comprises specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into SARS virus, gene product or fragment.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises and have immunocompetent antibody fragment (as Fab ' or (Fab)
2Fragment), heavy chain of antibody, light chain of antibody, chimeric antibody, humanized antibody etc.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the polypeptide of the present invention or the conjugate of purifying can be applied to animal to induce the generation of polyclonal antibody.For monoclonal antibody, can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).
Whether there are SARS virus or anti-SARS virus antibody in all available test sample of polypeptide of the present invention, conjugate and antibody, thereby as one of level of signification of early detection SARS.
Except being used for detecting, SARS specific polypeptide of the present invention also can be competed acceptor with virus in vivo, stops virus and film to merge, inhibition virus infection normal cell.In addition, polypeptide of the present invention and conjugate also can be used for preparing curative pharmaceutical composition or preventative vaccine composition.
Therefore, on the other hand, the present invention also provides a kind of composition, and it contains polypeptide of the present invention, conjugate or its composition of (a) safe and effective amount; And (b) pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol, adjuvant and combination thereof.Composition of the present invention can be prepared by ordinary method.In polypeptide of the present invention, its consumption for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
Major advantage of the present invention is:
(a) because the avidity height of SARS specific polypeptide of the present invention, polypeptide conjugate and anti-SARS virus antibody, the avidity height of antibody of the present invention and SARS virus, therefore, detection method sensitivity of the present invention, easy, accuracy rate is high.
(b) SARS specific polypeptide of the present invention also can be competed acceptor with virus in vivo, stops virus and film to merge, and inhibition virus infection normal cell is so the present invention has very high clinical value.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The synthetic of polypeptide
Press the aminoacid sequence shown in the table 1, on Peptide synthesizer, use synthetic polypeptide.It is synthetic after mass spectroscopy proves that the synthetic polypeptide conforms to design.
Table 1: amino acid sequence of polypeptide of the present invention
| Numbering: | Sequence | ??SEQ?ID?NO： |
| N albumen | ||
| N1-(21-44) | PTDSTDNNQNGGRNGARPKQRRPQ | ??????1 |
| N2-(138-160) | GALNTPKDHIGTRNPNNNAATVL | ??????2 |
| N3-(365-388) | PPTEPKKDKKKKTDEAQPLPQRQK | ??????3 |
Antigenic peptide and be connected (preparation of conjugate) carrier
For each polypeptide of embodiment 1 synthetic, dissolve (to some insoluble polypeptide with PBS earlier, can then make it molten entirely as if insoluble earlier with partly measuring the PBS dissolving with 1M NaOH adjusting PH), get 0.5ml polypeptide (concentration is respectively 0.5mg/ml, 1mg/ml) and 15mg BSA (1.5ml) mixing, add linking agent then (at the difference of crosslinked direction, select different linking agents, N-holds crosslinked adding glutaraldehyde, and the C-end adds EDAC), mix the back in room temperature reaction 5 hours, lucifuge is constantly rocked; After the crosslinked end, put into dialysis tubing, to 4 ℃ of dialysed overnight of PBS; The amount volume, with the BSA calculating concentration, standby in-20 ℃ of preservations.
Embodiment 3
The antigenic peptide immune animal prepares antibody:
Get the about 0.5ml of conjugate (conjugate content is respectively 0.1mg, 0.5mg) of embodiment 2 preparations, mix with CFA (complete Freund's adjuvant) after adding PBS 1.5ml, in Y-tube, push away repeatedly and beat, until the sensation effort, place after 3-5 minute, nothing two is separated and gets final product.
Will be through the antigen immune rabbit and the mouse of adjuvant emulsion processing; About every rabbit injections of antigens 3ml, mouse is only then injected 0.8ml/.Difference subcutaneous injection animal two sufficient pads, neck, back multi-point injection (every some 0.2ml).
Initial immunity is got same dosage polypeptide antigen, PBS after three weeks, mixes 2ml IFA (incomplete Freund's adjuvant), after the emulsification animal is carried out booster immunization, and injected dose is constant.
After three weeks animal is got blood, collect antiserum(antisera).Use the chromatography column of BSA bag quilt, remove antibody at BSA.
Select antibody titers antigens with higher polypeptide, the preparation monoclonal antibody.
Embodiment 4
Detect and use
(1) antigenic peptide detects patients serum's sample:
Use the antigenic peptide bag quilt of treated embodiment 1 preparation, with patients serum's hybrid reaction, result's antigen peptide section of the present invention can be discerned early stage specific antibody at SARS among the patients serum, and combination with it, and the washing back adds the HRP enzyme and connects two anti-reactions; Add chromogenic substrate at last, detect.
The result shows, polypeptide of the present invention can be specifically and the antibodies of anti-SARS virus.
(2) antibody (monoclonal antibody is with how anti-) detects pathogenic agent:
The antibody of the acquisition that obtains for embodiment 3 (how anti-and monoclonal antibody), with ELISA method and patients serum's reaction, the virus combination in detection tissue of patient liquid, the blood detects pathogenic agent.
The result shows that resist with monoclonal antibody of the present invention can combine with SARS virus specifically.
Embodiment 5
Immunoblot experiment
In the present embodiment, used antibody is respectively preimmune serum, N1-antiserum(antisera), N2-antiserum(antisera) and N3-antiserum(antisera) (antibody in the serum behind the immunize rabbit, not purifying).
The immunoblot experiment flow process is as follows:
1.SDS-PAGE：
Sample: the cell pyrolysis liquid that SARS virus infects
Applied sample amount: 100ul
Upper strata glue 3.5%, lower floor's glue 7%, upper strata glue 100V 1 hour, the glue 200V of lower floor 15 hours
2. commentaries on classics film:
Electric current: 1.0mA/cm2,2 hours
3. sealing:
5% skim-milk sealing 6 hours
4. hybridization
One is anti-, is diluted in TBS-Tween at 1: 100, and 4 degree hybridization are spent the night
Two anti-(goat-anti-rabbit-HRP is available from Santa Cruze companies): dilution in 1: 4000, hybridized 1 hour
Colour developing: ECL-Plus (available from Amersham company)
The result shows, the N protein binding of anti-N1, N2 and N3 antibodies specific ground and SARS virus.
Embodiment 6
Enzyme-linked immunosorbent assay (ELISA):
By 96 orifice plates, 4 ℃ are spent the night with BSA-N1, BSA-N2 cross-linking products bag.After confining liquid (3%Gelatin, 0.1% polysorbas20 the is dissolved in PBS) sealing, add the immunity back rabbit anteserum of doubling dilution, hatched 2 hours for 37 ℃, the washing back adds the IgG of the goat-anti rabbit of HRP mark, hatches 1 hour for 37 ℃, adds the substrate colour developing behind the thorough washing and measures 450nm place absorbance value.Gathering 9 routine clinical diagnosis patients SARS and 4 routine normal human serums, is that antigen is determined its IgG positive by the method for ELISA and immunofluorescence analysis with the employing virus cracking liquid.Antiserum(antisera) detects by the ELISA method the reactivity of synthetic peptide section (N1, N2).Roughly process is the same.Serum dilution is 1: 10, and the goat-anti people's of HRP mark IgG is anti-as two.All use patients serum's experiment all in the three-grade biological safety laboratory operation.
The result:
As table 2
Antibody titers in the rabbit anteserum after table 2 ELISA method mensuration peptide section N1, the N2 immunity
| Envelope antigen | ||||
| The peptide section | Serum sample | Neutralization | Peptide section-BSA | ????BSA |
| ????N1 | Preimmune serum | Do not have | ??4,000 | ????＜1,000 |
| Immunity back serum | ????BSA ????-8- | ??＞128,000 | ????1,000 | |
| Immunity back serum | Do not have | ??＞128,000 | ???128,000 | |
| ????N2 | Preimmune serum | Do not have | ??2,000 | ???＜1,000 |
| Immunity back serum | ????BSA | ??＞128,000 | ???1,000 | |
| Immunity back serum | Do not have | ??128,000 | ???16000 |
Embodiment 7
Peptide section N1 can combine with the IgG among the SARS patients serum
Since the peptide section N1 proteic good epi-position that is sars coronavirus N, can it combine with the IgG among the SARS patients serum so? in order to answer this problem, in the present embodiment, gather 9 routine clinical diagnosis patients SARS and 4 routine normal human serums, screen as antigen with peptide section N1, N2.
Found that patient 33%SARS that peptide section N1 is positive and all serum to the peptide section N2 reaction that is negative.Simultaneously, 4 routine normal human serums are to both be negative (table 3).
Table 3 ELISA method is measured in patient SARS and the normal human serum
At peptide section N1, the specific antibody test of N2
| Envelope antigen | Normal people's sequence number 1234 | Patient's SARS sequence number (clinical diagnosis) 123456789 |
| ?N1-BSA | ???-????-????-????- | ??+?????-?????-?????+?????-?????+?????-?????-?????- |
| ?N2-BSA | ???-????-????-????- | ??-?????-?????-?????-?????-?????-?????-?????-?????- |
Serum dilution: 1: 10
+: positive-: feminine gender
Embodiment 8
Sars coronavirus N albumen pronucleus expression and purifying:
In the present embodiment, with the proteic full-length gene of sars coronavirus N clone (Genbank accession number NC_004718) multiple clone site of (Qiagen company) to prokaryotic expression carrier pQE30.With recombinant plasmid transformed intestinal bacteria M15 abduction delivering albumen.Choose mono-clonal in corresponding resistance nutrient solution after the overnight incubation, be seeded in the nutrient solution of identical resistance shaking culture with 1: 10 ratio to OD
600Be 0.6～0.8, add IPTG to final concentration be 0.5mmol/L abduction delivering albumen.Behind the Ni-NTA affinitive layer purification, SDS-PAGE detects the N albumen of reorganization according to operational manual.
The Western trace:
N albumen behind the purifying behind 12% SDS-PAGE electrophoresis electrotransfer to pvdf membrane.After confining liquid (3%Gelatin) sealing, add immunity back rabbit anti-serum (1/1000 dilution), to hatch 1 hour for 37 ℃, the washing back adds the IgG (H+L) of the goat-anti rabbit of HRP mark, hatches 1 hour ECL colour developing behind the thorough washing for 37 ℃.
The result
The antibody that peptide section N1 induces generation has higher affinity to the sars coronavirus N albumen of prokaryotic expression
For verify antibody that peptide section N1, N2 induce generation whether can with sars coronavirus N protein binding, the N albumen (422 amino acid) of having expressed total length in the present embodiment with prokaryotic system is verified the power of its avidity by the method for Western trace.Albumen behind the purifying is through the SDS-PAGE electrophoresis, and the result is presented at the 47kD place a protein band (Fig. 3) clearly.
Rabbit anteserum has higher affinity to the sars coronavirus N albumen of prokaryotic expression after Western trace result (Fig. 4) the show peptide section N1 immunity, and N2 a little less than.And contrast GST (irrelevant albumen) and normal rabbit serum are all reactionless.These data show once more: peptide section N1 be the proteic good epi-position of sars coronavirus N and its induce generation antibody can with the N protein binding of expressing.
Discuss
The present invention has proved that first the proteic epi-position of sars coronavirus N (as N1 etc.) helps further to study the proteic function of N.
At first, from sars coronavirus N albumen, select two peptide section N1, N2 by computer software analysis.In theory, both all have antigenicity preferably based on its antigenicity index.The titre analysis of antibody the rabbit anti-serum after immunity, both are similar.
Secondly, the N albumen whether synthetic peptide section of the present invention can simulate native state is the key that the peptide section is used.The antibody that experimental result show peptide section N1 induces generation has higher affinity to the sars coronavirus N albumen of prokaryotic expression, and prompting peptide section N1 is the proteic good epi-position of sars coronavirus N.And peptide section N1 induces the antibody of generation will help the proteic research to N.In addition, only can induce the protein binding that produces high titer antibody and this antibody capable and native state with synthetic peptide section.
In order further to confirm that two peptide sections induce the antibody of generation to reflect their immunogenic similarities and differences in vivo whether really with combining of expressing protein, the method for the inventor by ELISA detected two kinds of peptide sections and SARS patients serum's reactivity.The result shows that peptide section N1 is that an energy is induced the good epitope that produces antibody in the SARS patient body.Although have only 33% patient to be positive, can explain.Also have only 40% clinical diagnosis patient SARS to be positive because detect as antigen with complete N albumen.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉sars coronavirus related polypeptide and application thereof
<130>033332
<150>CN?03128831.6
<151>2003-05-26
<160>3
<170>PatentIn?version?3.1
<210>1
<211>24
<212>PRT
＜213〉SARS virus (SARS virus)
<400>1
Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly?Arg?Asn?Gly?Ala
1???????????????5???????????????????10??????????????????15
Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln
20
<210>2
<211>23
<212>PRT
＜213〉SARS virus (SARS virus)
<400>2
Gly?Ala?Leu?Asn?Thr?Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn
1???????????????5???????????????????10??????????????????15
Asn?Asn?Ala?Ala?Thr?Val?Leu
20
<210>3
<211>24
<212>PRT
＜213〉SARS virus (SARS virus)
<400>3
Pro?Pro?Thr?Glu?Pro?Lys?Lys?Asp?Lys?Lys?Lys?Lys?Thr?Asp?Glu?Ala
1???????????????5???????????????????10??????????????????15
Gln?Pro?Leu?Pro?Gln?Arg?Gln?Lys
20
Claims (10)
1. a peptide species is characterized in that, its aminoacid sequence is SEQ ID NO:1,2 or 3.
2. a peptide species conjugate is characterized in that, described conjugate is made of polypeptide, linking agent and BSA, and wherein said amino acid sequence of polypeptide is SEQ ID NO:1,2 or 3, and described linking agent is selected from glutaraldehyde or EDAC.
3. an antibody is characterized in that, described antibodies specific ground combines with the described polypeptide of claim 1.
4. antibody as claimed in claim 3 is characterized in that described antibody is monoclonal antibody.
5. a detection kit is characterized in that, it contains the described polypeptide of claim 1, the described polypeptide conjugate of claim 2, the described antibody of claim 3 or its combination.
6. whether there is the method for SARS virus or anti-SARS virus antibody in the vitro detection sample, it is characterized in that, comprise step:
(a) sample is contacted with the material that is selected from down group: the described polypeptide of claim 1, the described polypeptide conjugate of claim 2, the described antibody of claim 3 or its combination;
(b) detect whether form antigen-antibody complex, wherein form mixture and just represent to exist in the sample SARS virus or anti-SARS virus antibody.
7. method as claimed in claim 6 is characterized in that, described sample is serum or sputum.
8. method as claimed in claim 6 is characterized in that, described material is that aminoacid sequence is SEQID NO:1,2 or 3 polypeptide.
9. method as claimed in claim 6 is characterized in that, described material is the described antibody of claim 3, and described antibody is monoclonal antibody.
10. method as claimed in claim 6 is characterized in that, the detection method of step (b) is a fluorescence detection.
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| CNB031292895A CN100467485C (en) | 2003-05-26 | 2003-06-13 | SARS coronary virus related polypeptide and its uses |
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|---|---|---|---|
| CN03128831.6 | 2003-05-26 | ||
| CN03128831 | 2003-05-26 | ||
| CNB031292895A CN100467485C (en) | 2003-05-26 | 2003-06-13 | SARS coronary virus related polypeptide and its uses |
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| CN1572798A true CN1572798A (en) | 2005-02-02 |
| CN100467485C CN100467485C (en) | 2009-03-11 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100588660C (en) * | 2003-06-25 | 2010-02-10 | 中国科学院上海生命科学研究院 | Antigenic determinant of SARS coronavirus nucleocapsid protein and application thereof |
| CN1903878B (en) * | 2005-07-26 | 2011-09-07 | 复旦大学 | Anti SARS virus human source nature antibody IgG Fab section |
| CN111381049A (en) * | 2020-03-30 | 2020-07-07 | 天津纽赛生物技术有限公司 | Serology type detection test paper and detection method for rapid new coronavirus antibody |
| CN111690043A (en) * | 2020-05-22 | 2020-09-22 | 秦小波 | NTD polypeptide based on SARS-CoV-2 nucleoprotein, coding gene, recombinant vector, expression method and application |
| CN111978378A (en) * | 2020-08-10 | 2020-11-24 | 武汉大学 | SARS-CoV-2 antigen polypeptide and its application |
-
2003
- 2003-06-13 CN CNB031292895A patent/CN100467485C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100588660C (en) * | 2003-06-25 | 2010-02-10 | 中国科学院上海生命科学研究院 | Antigenic determinant of SARS coronavirus nucleocapsid protein and application thereof |
| CN1903878B (en) * | 2005-07-26 | 2011-09-07 | 复旦大学 | Anti SARS virus human source nature antibody IgG Fab section |
| CN111381049A (en) * | 2020-03-30 | 2020-07-07 | 天津纽赛生物技术有限公司 | Serology type detection test paper and detection method for rapid new coronavirus antibody |
| CN111690043A (en) * | 2020-05-22 | 2020-09-22 | 秦小波 | NTD polypeptide based on SARS-CoV-2 nucleoprotein, coding gene, recombinant vector, expression method and application |
| CN111690043B (en) * | 2020-05-22 | 2022-12-02 | 秦小波 | NTD polypeptide based on SARS-CoV-2 nucleoprotein, coding gene, recombinant vector, expression method and application |
| CN111978378A (en) * | 2020-08-10 | 2020-11-24 | 武汉大学 | SARS-CoV-2 antigen polypeptide and its application |
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