CN1552729A - SARS early diagnostic method and reagent box - Google Patents
SARS early diagnostic method and reagent box Download PDFInfo
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- CN1552729A CN1552729A CNA031288324A CN03128832A CN1552729A CN 1552729 A CN1552729 A CN 1552729A CN A031288324 A CNA031288324 A CN A031288324A CN 03128832 A CN03128832 A CN 03128832A CN 1552729 A CN1552729 A CN 1552729A
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Abstract
A method and reagent kit for the early diagnosis of SARS features that the polypeptide as antigen synthesized artificially from the S protein of SARS virus and the antibody generated by the animal immunized by said antigen is used in enzyme-linked immunoassay to detect the early SARS antibody IgM or IgG or the SARS pathogen from human serum specimen.
Description
Technical field
The present invention relates to method of early diagnosis and the test kit of SARS.Particularly, the present invention uses artificial synthetic to be derived from the proteic polypeptide of SARS virus S, 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:1,2,3,4,5,6,7,8,9 or 10.These SARS specific polypeptides can be specifically and the antibodies of anti-SARS virus.
In a second aspect of the present invention, one 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:1,2,3,4,5,6,7,8,9 or 10, 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:1,2,3,4,5,6,7,8,9 or 10 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 that the preimmune serum with the S6 correspondence resists the result who carries out immunoblotting as one.
Fig. 4 has shown with the S6-antiserum(antisera) as an anti-result who carries out immunoblotting.
Fig. 5 has shown with the S10-antiserum(antisera) as an anti-result who carries out immunoblotting.
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, reaches 1200 aminoacid sequences from S albumen like this to filter out following a plurality of potential antigen sites (table 1).
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) 2 fragments), 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： |
| S albumen | ||
| S1-(34-54) | | 1 |
| S2-(94-113) | KSNVVRGWVFGSTMNNKSQS | 2 |
| S3-(197-216) | YKGYQPIDVVRDLPSGFNTL | 3 |
| S4-(273-293) | TDAVDCSQNPLAELKCSVKSF | 4 |
| S5-(297-316) | KGIYQTSNFRVVPSGDVVRF | 5 |
| S6-(332-351) | TKFPSVYAWERKKISNCVAD | 6 |
| S7-(436-455) | YNYKYRYLRHGKLRPFERDI | 7 |
| S8-(540-559) | PSSKRFQPFQQFGRDVSDFT | 8 |
| S9-(731-753) | CANLLLQYGSFCTQLNRALSGIA | 9 |
| S10-(758-780) | RNTREVFAQVKQMYKTPTLKYFG | 10 |
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 each 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 the preimmune serum of S6 correspondence, S6-antiserum(antisera) and S10-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: 20ul
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 room temperature hybridization 1 hour at 1: 500
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 is shown in Fig. 3-5.Wherein Fig. 3 has shown that the preimmune serum with the S6 correspondence resists the result who carries out immunoblotting as one.Fig. 4 has shown with the S6-antiserum(antisera) as an anti-result who carries out immunoblotting.Fig. 5 has shown with the S10-antiserum(antisera) as an anti-result who carries out immunoblotting.Four arrows in three figure left sides are standard molecular weight, are respectively from top to bottom: 175Kd, 83Kd, 62Kd, 47Kd.Right side arrow prompts for S albumen.
From Fig. 3-5 as can be seen, these two antibody of S6 and S10 all have a tangible band near 180Kd, and anti-not having for preimmune serum (being equivalent to negative control).Difference stripe size and S protein seemingly, prompting right side arrow indication place band is a S albumen.This shows, the S protein binding of anti-S6 and S10 antibodies specific ground and SARS virus.In addition, since at other position and preimmune serum than not finding notable difference, so point out S albumen in SARS virus, not shear.
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 method of early diagnosis and test kit
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＜213〉SARS virus (SARS virus)
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Thr?Ser?Ser?Met?Arg?Gly?Val?Tyr?Tyr?Pro?Asp?Glu?Ile?Phe?Arg?Ser
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＜213〉SARS virus (SARS virus)
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Lys?Ser?Asn?Val?Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met?Asn?Asn
1???????????????5???????????????????10?????????????????15
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<210>3
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＜213〉SARS virus (SARS virus)
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Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu?Pro?Ser?Gly
1???????????????5???????????????????10??????????????????15
Phe?Asn?Thr?Leu
20
<210>4
<211>21
<212>PRT
＜213〉SARS virus (SARS virus)
<400>4
Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys
1???????????????5???????????????????10??????????????????15
Ser?Val?Lys?Ser?Phe
20
<210>5
<211>20
<212>PRT
＜213〉SARS virus (SARS virus)
<400>5
Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp
1???????????????5???????????????????10??????????????????15
Val?Val?Arg?Phe
20
<210>6
<211>20
<212>PRT
＜213〉SARS virus (SARS virus)
<400>6
Thr?Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn
1???????????????5???????????????????10??????????????????15
Cys?Val?Ala?Asp
20
<210>7
<211>20
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＜213〉SARS virus (SARS virus)
<400>7
Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg?Pro?Phe
1???????????????5???????????????????10??????????????????15
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20
<210>8
<211>20
<212>PRT
＜213〉SARS virus (SARS virus)
<400>8
Pro?Ser?Ser?Lys?Arg?Phe?Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val
1???????????????5???????????????????10??????????????????15
Ser?Asp?Phe?Thr
20
<210>9
<211>23
<212>PRT
＜213〉SARS virus (SARS virus)
<400>9
Cys?Ala?Asn?Leu?Leu?Leu?Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln?Leu?Asn
1???????????????5???????????????????10??????????????????15
Arg?Ala?Leu?Ser?Gly?Ile?Ala
20
<210>10
<211>23
<212>PRT
＜213〉SARS virus (SARS virus)
<400>10
Arg?Asn?Thr?Arg?Glu?Val?Phe?Ala?Gln?Val?Lys?Gln?Met?Tyr?Lys?Thr
1???????????????5???????????????????10??????????????????15
Pro?Thr?Leu?Lys?Tyr?Phe?Gly
20
Claims (10)
1. a peptide species is characterized in that, its aminoacid sequence is SEQ ID NO:1,2,3,4,5,6,7,8,9 or 10.
2. a peptide species conjugate, it 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:1,2,3,4,5,6,7,8,9 or 10, 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:1,2,3,4,5,6,7,8,9 or 10 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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| CNB031288324A CN100467484C (en) | 2003-05-26 | 2003-05-26 | SARS early diagnostic method and reagent box |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof |
| CN111289745A (en) * | 2020-03-25 | 2020-06-16 | 中山生物工程有限公司 | Novel coronavirus IgM antibody enzyme-linked immunoassay kit and detection method thereof |
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2003
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof |
| CN111289745A (en) * | 2020-03-25 | 2020-06-16 | 中山生物工程有限公司 | Novel coronavirus IgM antibody enzyme-linked immunoassay kit and detection method thereof |
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| CN100467484C (en) | 2009-03-11 |
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