CN1796565A - Recombined virus for expressing spike protein of SARS coronavirus and preparation method - Google Patents
Recombined virus for expressing spike protein of SARS coronavirus and preparation method Download PDFInfo
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Abstract
This invention publishes a recombined autographa Californica multicapsid nucleopolyhedrovirus (AcMNPV) which can express the spike protein of SARS coronavirus in pestle cells. This recombined virus, with its conservation number CGMCC No.1280, is prepared as follows: the spike protein of SARS coronavirus is subcloned to pFastBac1 so as to construct transfer vector pFastBac1-SS which is subsequently used to transform DH10Bac, with the target product of positive recombined virus. It has been proved by experiments that the recombined virus obtained in this invention is able to express spike protein of SARS coronavirus efficiently in pestle cells, and the sensitivity of the expressed spike protein which serves as coating antigen for human SARS-CoV positive serum testing is no less than that of the whole SARS-CoV virus.
Description
Technical field
The present invention relates to a kind of recombinant virus, relate in particular to a kind of can be at kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus of expressed in insect cells sars coronavirus spike protein and its production and application.
Background technology
(severe acute respiratory syndrome SARS) is emerging human virus's sexually transmitted disease to severe acute respiratory syndrome.The human susceptibility that SARS-CoV (sars coronavirus) is had height.A large amount of SARS-CoV the infecteds show as stealthy the infection or the property crossed an atypical pneumonia symptom.Simultaneously, there is some evidence that animal, particularly wildlife may be the storage hosts naturally of this virus or propagate the host.Carry out extensive, tight serosurvey, to epidemiological surveillance, investigate thoroughly nature or propagate host SARS-CoV source significant.
The operation of SARS-CoV is subjected to strict Biosafety to require restriction.It is significant as detecting antigen to adopt the recombinant virus structural protein to replace the cell cultures totivirus.Sars coronavirus spike protein (SARS-CoV S) is the main structural protein of sars coronavirus, the process of invading host cell with SARS-CoV is closely related, be the major antigen albumen of SARS-CoV, immunity of energy inducing cell and humoral immunization (XiaoDong WU, Bo SHANG, et al.The Spike Protein of Severe AcuteRespiratory Syndrome (SARS) Is Cleaved in Virus Infected Vero-E6cells.Cell Res, 2004Sep 26:1～27; Qin ED.et al.A complete sequence and comparative analysisofa SARS-associated virus.Chin.Sci.Mull.2003.48:941-948; Lv Yanbo waits the B cell epitope prediction of .SARS virus S protein. Third Military Medical University's journal, 2004,26 (2): 101-103.)。The S genetic comparison of SARS-CoV is conservative, and there are a plurality of B cell dominant antigen epi-positions in S albumen, adopt E.coli to express S1, though being proved to be, S2 albumen has antigenicity (Ho-sheng Wu preferably, Shu-Chunchiu, Tsan-chang Tseng.Serologic and Molecular biologic methods forSARS-associated coronavirus infection, Taiwan.Emerging infectious diseases.2004.Vol.10.No.2.February.), but this method exists processing modification after the expression product translation and space conformation and SARS-CoV difference bigger simultaneously, preparation process is loaded down with trivial details, exists many loaded down with trivial details and disadvantageous sex change conditions consequently to be unfavorable for keeping defectives such as the proteic native configurations of S in the affinity purification process.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of can be at the kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus of expressed in insect cells SARS-CoV spike protein.
The preserving number of this kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus (hvri-bh-bsn) is CGMCCNo.1280, the preservation time: on December 27th, 2004, preservation place: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
Another technical problem to be solved by this invention provides the preparation method of this kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
SARS-CoV S gene (SS) subclone to pFastBacl, is made up transfer vector pFastBacl-SS, continue to transform DH
10Bac, the positive recombinant virus of picking, the positive recombinant virus of picking promptly gets kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus of the present invention.
Another technical problem that the present invention also will solve provides a kind of reorganization sars coronavirus spike protein.
A kind of reorganization sars coronavirus spike protein is prepared from by following method:
With kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus CGMCC No.1280 transfection insect cell of the present invention,, get supernatant promptly with the insect cell cracking of being infected.
Recombinant baculovirus of the present invention, can highly effectively expressing SARS-CoV S albumen behind the infected insect cell, the anti-patient's positive serum of crossing of expressed reorganization SARS-CoV S albumen and deactivation SARS-CoV totivirus hyper-immune serum and infection all has good immune response originality, has confirmed that further S albumen is one of SARS-CoV main immunogens structural protein.
The reorganization SARS-CoV S albumen (rSS) that the present invention is expressed is as the ELISA envelope antigen, compare with VeroE6 growth totivirus envelope antigen, not only preparation process is not subjected to strict P3+ Biosafety condition restriction, and has better susceptibility and specificity; With the E.coli prokaryotic expression SARS-CoV reorganization S1 that has reported, S2 albumen is compared, not only processing modification after the expression product translation and space conformation and SARS-CoV are more approaching, and preparation process is simplified, only needing to add PBS ultrasonic degradation recombinant virus-infected cell gets final product, avoid the loaded down with trivial details and disadvantageous sex change condition in the affinity purification process, helped keeping the native configurations of rSS.
The sf9 cell of expressing rSS is used for IFA, and rapid detection blood serum special antibody response shows as good sensitivity and specificity equally, demonstrates good prospects for application.
In a word, the present invention's SARS-CoV S albumen of recombinating has advantages such as immune response originality is good, preparation is easy, the source is abundant, can substitute the SARS-CoV totivirus antigen of VeroE6 cells produce, preparation safety economy, special ELISA and the IFA diagnostic reagent of sensitivity are applied to detect the epidemiological surveillance of animal or human's class SARS-CoV specific antibody.
Description of drawings
Fig. 1 rSS Western-Blot detects.
1: normal sf9 cell lysate supernatant 2: the sf9 cell lysate supernatant 3,4 that infects wild-type baculovirus: the sf9 cell lysate supernatant that infects rBac-SS.
Fig. 2 rSS detects the anti-deactivation SARS-CoV of chicken hyper-immune serum fluorescent signal scanning result as antigen I FA.
Fig. 3 rSS is that special serum I gY antibody of the anti-SARS-CoVN albumen of indirect ELISA Detection of antigen chicken and SARS-CoV infect the anti-special IgG antibody test of the patients serum's S albumen result that crosses.
Further describe beneficial effect of the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment
Preparation of [embodiment] recombinant baculovirus of the present invention and the expression in insect cell
One, test materials
Plasmid pFastBacl and DH
10BacAvailable from Invitrogen company; SARS-CoV S protein gene cDNA cloned plasmids pBSS ((preservation of inventor laboratory)), sf9 insect cell (preservation of inventor laboratory).The anti-SARS-CoV height of chicken is exempted from positive serum and is prepared from via the immune repeatedly SPF chicken of SARS-CoV BJol strain Vero E6 cell cultures cracking supernatant.
Two, test method
With S gene subclone to pFastBacl (seeing Invitrogen Bac-to-Bac BaculovirusExpression Systems specification sheets).Enzyme after cutting and identifying it is transformed with PCR (with reference to the preparation method of following document: Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi. molecular cloning experiment guide (second edition) [M]. Beijing: Science Press, 1993,26:674-683.) to DH
10Bac, PCR screen positive recombinant clone Bacmid-SS J (with reference to the preparation method of following document: Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi. molecular cloning experiment guide (second edition) [M]. Beijing: Science Press, 1993,26:674-683.).Prepare the positive recombinant clone DNA (preparation method of the following document of reference: Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi. molecular cloning experiment guide (second edition) [M]. Beijing: Science Press, 1993,26:674-683.), transfection sf9 cell (seeing Invitrogen transfection agents specification sheets), treat to carry out recombinant virus (rBac-SS) plaque purification and virus amplification (Cho-Hua Wan after the cytopathy positive, Michael I, Riley, Reuel R.et al.Expression of Sendai Virus Nucleocapsid Protein ina Baculovirus expression system and application to diagnostic assays for sendaivirus infection.Journal of Clinical.Microbiology, Auguest, 1995, p.2007-11.), Proteinase K-SDS-phenol/chloroform extracting virus genom DNA, synthetic following primer: ss-f:5 ' agag
AagcttTattgaggacttgctct3 ' and ss-r:5 ' gg
CtcgcgagTtatgtgtaatgtaatttg 3 ' (underscore is respectively HindIII and XhoI site), with ss-f, ss-r, M13+ and M13-is primer PCR checking reorganization positive-virus, the reorganization positive-virus is kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus of the present invention (rBac-SS), and 4 ℃ of preservations of kind poison are standby.
RBac-SS kind poison infects the sf9 cell by 1: 10 volume ratio, the centrifugal results cells infected of 1000g after 72 hours, and PBS centrifuge washing twice, resuspended by 10: 1 usefulness PBS, ultrasonic treatment cell after three freeze thawing, centrifugal back results supernatant carries out SDS-PAGE with supernatant.Supernatant is carried out SDS-PAGE with 12% separation gel, coomassie brilliant blue staining, estimation S albumen relative expression level; Measure the cell lysate total protein content, calculate the S protein content.Simultaneously, cell lysate SDS-PAGE electricity is transferred to nylon membrane (Ameresco), the sealing of 5% skimming milk, PBST (0.05%Tween20) washing, it is one anti-that the anti-SARS-CoV height of 1: 500 times of PBST dilution chicken is exempted from positive serum, 1: 10000 times of PBST dilution horseradish peroxidase (HRP) mark anti-chicken IgG of rabbit (Sigma) is two anti-, and the DAB colour developing stopped with deionized water after 3～5 minutes.
The result: the about 180-200KD of rBac-SS cells infected exogenous protein expression product, size conforms to desired value.It is one anti-exempting from positive serum with the anti-SARS-CoV height of chicken, Western-Blot demonstrates special 180-200KD and detects albumen (Fig. 1), presentation of results SARS-CoV S protein gene obtains to express in baculovirus rBac-SS, and reorganization SARS-CoV S albumen (rSS) expression amount accounts for full total protein of cell about 10%.
Testing routine 1rSS is the special serum I gY antibody of the anti-SARS-CoV S of indirect ELISA Detection of antigen chicken albumen
One, test materials
1, for test agent: the rSS that the embodiment of the invention 1 is prepared.
2, the anti-SARS-CoV height of chicken is exempted from positive serum via the immune repeatedly SPF chicken preparation of SARS-CoV BJol strain Vero E6 cell cultures cracking supernatant, and veterinary institute preparation in Harbin provides.
Two, test method and result
Use NaHCO
3(pH=9.6) the rBac-SS infection sf9 cell lysate of 100 times of dilutions of damping fluid embodiment 1 prepares antigen, and by every hole 100ul, promptly recombinant s protein antigen amount is every hole 0.1ug, and 4 ℃ of bags are spent the night by 96 hole elisa plates (Nunc).It is one anti-that positive serum is exempted from 5% skimming milk PBST (0.05%Tween20) sealing, 1: 500 anti-SARS-CoV height of dilution chicken, and other establishes the negative contrast of identical extension rate SPF chicken serum; 1: 10000 times of PBST dilution HPR mark anti-chicken IgG of rabbit (Sigma) is two anti-, the PBST washing, substrate OPD and TMB (Sigma) act on 25min, after the 2M sulfuric acid termination reaction, measure OD value (Bio-Rad, Benchmark plus), each serum dilution is established 3 parallel holes at least, averages and calculates the P/N value.
Test-results: bag is by rSS 0.1ug, and the 500 times of anti-deactivation SARS-CoV of dilution chicken serum are tested one anti-OD
490nmMean value is the OD of 1.323,500 times of dilution SPF chicken control serums
490nmMean value is 0.04, and the P/N value is that 33.08 (Fig. 3 a).
The test-results explanation, the expressed sars coronavirus spike protein of recombinant virus of the present invention has good immune response originality as the indirect ELISA envelope antigen.
Testing routine 2rSS is that indirect ELISA Detection of antigen SARS-CoV infects the anti-special IgG antibody of patients serum's S albumen of crossing
For test agent: the reorganization sars coronavirus nucleoprotein (rSS) that the embodiment of the invention 1 is prepared.
Test materials: diluted the anti-patient's positive serum excessively of SARS-CoV infection and healthy people's negative control sera from the special IgG antibody assay kit of SARS-CoV (traditional Chinese medicines examination word S20030004, Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C and Beijing Institute of Gene Science, Chinese Academy of Sciences's development)
Test method and result:
The elisa plate of SARS-CoV bag quilt detects the special IgG antibody of people's anti-SARS-CoV rehabilitation patient positive serum to be undertaken by the test kit specification sheets, and the anti-human IgG of HRP mark is two anti-, and TMB is the substrate colour developing, measures OD
450nmValue.Concrete grammar is as follows:
Use NaHCO
3(pH=9.6) the above-mentioned rBac-SS infection of 100 times of dilutions of damping fluid sf9 cell lysate prepares antigen, and by every hole 100ul, promptly recombinant s protein antigen amount is every hole 0.1ug, and 4 ℃ of bags are spent the night by 96 hole elisa plates (Nunc).5% skimming milk PBST (0.05%Tween20) sealing, to infect the anti-patient of mistake positive serum be one anti-with diluting SARS-CoV, other establishes the negative contrast of identical extension rate health human serum; The anti-human IgG of 1: 10000 times of PBST dilution HRP enzyme labelling is two anti-, the PBST washing, substrate OPD and TMB (Sigma) act on 25min, after the 2M sulfuric acid termination reaction, measure OD value (Bio-Rad, Benchmark plus), each serum dilution is established 3 parallel holes at least, averages and calculates the P/N value.
Test-results: 0.1ug rSS bag is by the positive tested serum OD in hole
450nmAverage 1.015, the tested serum OD of healthy people's negative control
450nmAverage is 0.01 (Fig. 3 B); SARS-CoV totivirus bag is by the positive tested serum OD in hole
450nmAverage is 0.52, negative control sera OD
450nmAverage is 0.01 (Fig. 3 C); 0.1ug rSS and two kinds of envelope antigen P/N of SARS-CoV totivirus value are respectively 20.3 and 10.4 (negative control value be 0.01 calculate by 0.05).The cutoff value that provides in the test kit is the OD of 0.13+ negative control sera
450nmAverage.RSS is envelope antigen OD
450nmValue is all greater than the cutoff value.
Test-results shows, rSS is better than SARS-CoV totivirus envelope antigen as the susceptibility that envelope antigen is used to detect human SARS-CoV positive serum.
Test routine 3rSS and detect SARS-CoV antibody as antigen I FA
Test materials: the anti-SARS-CoV height of chicken is exempted from positive serum via the immune repeatedly SPF chicken preparation of SARS-CoV BJol strain Vero E6 cell cultures cracking supernatant, and veterinary institute preparation in Harbin provides;
Test method and result
RBac-ss is infected resuspended back, sf9 cell PBS washing back drip on slide, dry back 70% methyl alcohol naturally and fix.Be one anti-with the SPF chicken negative serum of 1: 100 dilution the chicken anti-SARS-CoV high property exempted from serum and identical extension rate respectively.PBST washing back adds two of 1: 5000 dilution fluorescein (FITC) mark and resists, effect 30min, PBST washing back fluorescent microscope (Leica DMIRES2) observations.
Test-results: the anti-hyper-immune serum of chicken shows strong positive fluorescent signal (Fig. 2 A), and 1: 100 dilution SPF chicken control serum is fluorescent signal feminine gender (Fig. 2 B) then.Illustrate that the present invention makes the expressed sars coronavirus spike protein of recombinant virus and is used for IFA and detects and to have good sensitivity and specificity.
Claims (8)
1, a kind of can be at kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus (hvri-bh-bsn) CGMCC of expressed in insect cells sars coronavirus spike protein No.1280.
2, with the described recombinant virus transformed host cells of claim 1.
3, according to the described host cell of claim 2, described host cell is an insect cell.
4, according to the described host cell of claim 4, described insect cell is the sf9 cell.
5, the method for the kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus CGMCC No.1280 of preparation claim 1, step is as follows:
1) with sars coronavirus spike protein gene subclone to pFastBacl, make up transfer vector pFastBacl-SS,
2) transfer vector with the step 1) gained transforms DH
10Bac, the positive recombinant virus of picking promptly.
6, a kind of reorganization sars coronavirus spike protein is prepared from by following method:
With the kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus CGMCC No.1280 transfection insect cell of claim 1,, get supernatant promptly with the insect cell cracking of being infected.
7, in accordance with the method for claim 6, the method for described cracking insect cell is a ultrasonic degradation.
8, the purposes of the kernel polyhedra of alfalfa silver mosquito noctuid recombinant virus CGMCC No.1280 of claim 1 in the epidemiological surveillance medicine of preparation animal or human class SARS-CoV specific antibody.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101100680B (en) * | 2007-06-15 | 2010-07-21 | 中国科学院武汉病毒研究所 | Recombination baculoviral for highly effectively expressing SARS coronavirus S protein and construction thereof |
| CN101948814A (en) * | 2010-08-26 | 2011-01-19 | 国家兽用生物制品工程技术研究中心 | Recombinant baculovirus for expressing spike protein of chicken infectious bronchitis virus and application thereof |
| CN111606981A (en) * | 2020-05-27 | 2020-09-01 | 中国医学科学院基础医学研究所 | SARS-COV coronavirus S1 protein polypeptide and its application |
| CN113388010A (en) * | 2020-03-11 | 2021-09-14 | 洛阳普泰生物技术有限公司 | Novel coronavirus recombinant protein S1 antigen and double-antigen sandwich ELISA antibody detection kit |
-
2004
- 2004-12-30 CN CN 200410102943 patent/CN1796565A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101100680B (en) * | 2007-06-15 | 2010-07-21 | 中国科学院武汉病毒研究所 | Recombination baculoviral for highly effectively expressing SARS coronavirus S protein and construction thereof |
| CN101948814A (en) * | 2010-08-26 | 2011-01-19 | 国家兽用生物制品工程技术研究中心 | Recombinant baculovirus for expressing spike protein of chicken infectious bronchitis virus and application thereof |
| CN113388010A (en) * | 2020-03-11 | 2021-09-14 | 洛阳普泰生物技术有限公司 | Novel coronavirus recombinant protein S1 antigen and double-antigen sandwich ELISA antibody detection kit |
| CN113388010B (en) * | 2020-03-11 | 2022-09-13 | 洛阳普泰生物技术有限公司 | Novel coronavirus recombinant protein S1 antigen and double-antigen sandwich ELISA antibody detection kit |
| CN111606981A (en) * | 2020-05-27 | 2020-09-01 | 中国医学科学院基础医学研究所 | SARS-COV coronavirus S1 protein polypeptide and its application |
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