CN104211786A - Bovine rotavirus recombinant VP7 protein antigen and preparation method thereof - Google Patents
Bovine rotavirus recombinant VP7 protein antigen and preparation method thereof Download PDFInfo
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Abstract
The invention provides a bovine rotavirus recombinant VP7 protein antigen and a preparation method thereof and relates to a protein antigen and the preparation method thereof, and the bovine rotavirus recombinant VP7 protein antigen can be used for solving the problems that the existing bovine rotavirus detection method cannot simultaneously meet the requirements of equipment and operation simplification, good specificity and large-scale clinical popularization. The bovine rotavirus recombinant VP7 protein antigen is coded by 644bp basic groups and has a size of 40kDa. The preparation method comprises the following steps: 1, extracting genome RNA of a bovine rotavirus BRV-DQ strain; 2, carrying out reverse transcription, PCR (Polymerase Chain Reaction) amplification and purification on the genome RNA and then connecting the genome RNA with a pMD18-T carrier so as to obtain pMD18-T-VP7 plasmids; 3, simultaneously carrying out enzyme digestion on pMD18-T-VP7 and pET30a, connecting and converting into host bacteria so as to obtain recombinant bacteria pET30a-VP7/BL21; 4, carrying out inducible expression on recombinant protein; and 5, purifying the recombinant protein so as to obtain the bovine rotavirus recombinant VP7 protein antigen. The bovine rotavirus recombinant VP7 protein antigen is applied to the field of detection of bovine rotaviruses.
Description
Technical field
The present invention relates to a kind of proteantigen and preparation method thereof.
Background technology
Bovine rota (Rotavirus, RV) belongs to Reoviridae (Reoviridae) rotavirus (Rotavirus), is one of main pathogen of calf Non-bacterial diarrhea.Bovine rota is found and proves the cause of disease of newborn calf diarrhoea, called after NCDV strain (NebraskacalfdiarrheaVirus, NCDV) first in nineteen sixty-eight by people such as Mebus.This virus can cause that animal spirit is depressed, watery diarrhea, oxypathy and appetite absolutely useless, infect 15-45 age in days calf more.1980, China detected rotavirus first in buffalo, ox diarrhoea ight soil, proves that this virus is in the existence of China.Also there is subsequently the report of a large amount of calf viral diarrheas.Bovine rota is popular extensively, sickness rate is high and calf is had to larger harm, and oneself becomes a kind of worldwide disease.If secondary Other diseases, mortality ratio can increase greatly, to cattle-raising, has caused huge financial loss.
The traditional diagnosis method of bovine rota is as cell isolation method, and due to except A type bovine rota, many strains of other bovine rota fail to adapt to cell cultures so far, and separated success ratio is about 40%-70%.Due to this method complex operation, need special culture condition and technique, separation cycle is longer, expense is higher, therefore and be not suitable as clinical detection and the diagnostic method of wide model; Electron microscopy, though this method is for detecting " gold standard " of this cause of disease, but because the required requirement of experiment of electron microscopy more also needs Special Equipment and the talent, and be not suitable for being applied to extensive sample detection, and whether virus particle is broken in ight soil, whether form is completely or whether quantity enough etc. that aspect all can affect Electronic Speculum result; Modern Molecular Detection means are as polyacrylamide gel electrophoresis (PAGE), and the method high specificity, not only can be used for detecting bovine rota, but also can differentiate the population of bovine rota.PAGE, as BRV authentication method, does not need special equipment and simple to operate, when can be used for a large amount of sample, detects.Its deficiency more easily by the RNA enzyme liberating in air or medicine, makes result inaccurate mainly due to viral RNA, occurs false negative; The PCR detection method of BRV classics is reverse transcription PCR (RT-PCR), 2 sections of nucleotides sequences that each type of ox A rotavirus Chang Yiqi structural protein VP7 is all guarded are classified primer as, fast, special differentiates virus, but leaching process complexity and easily pollution due to RNA, be more suitable for laboratory diagnosis, for large-scale clinical universal, still difficult; Serology test is undoubtedly diagnostic means comparatively desirable in clinical detection, wherein ELISA diagnostic method is without expensive plant and instrument, cost is low, susceptibility and specificity are high, the advantages such as no radioactivity pollute, be easy to promote in basic unit, and the indirect ELISA method of setting up to express capsid protein has again the advantage that overcomes antigen toxin expelling, loose malicious potentially dangerous.
Summary of the invention
The present invention, in order to overcome the problems referred to above, provides a kind of bovine rota recombinant VP7 protein antigen antigen and preparation method thereof.
Bovine rota recombinant VP7 protein antigen antigen of the present invention is by 644bp alkali yl coding, and size is 40kDa, and base sequence is as shown in SEQ ID NO:1.
The preparation method of bovine rota recombinant VP7 protein antigen antigen of the present invention, carries out according to the following steps:
One, extract the geneome RNA of bovine rota BRV-DQ strain; Two, RNA reverse transcription step 1 being obtained is cDNA, uses upstream primer P1 and downstream primer P2 to carry out pcr amplification to cDNA, and PCR product is connected with pMD18-T carrier after glue reclaims purifying, obtains pMD18-T-VP7 plasmid; Three, pMD18-T-VP7 plasmid and expression vector pET30a are all adopted to Nco I and Hind III double digestion, then by T4DNA ligase enzyme, connect, connect product and proceed to Host Strains BL21, obtain recombinant bacterium pET30a-VP7/BL21; Four, the abduction delivering of recombinant protein; Five, the purifying of recombinant protein, obtains bovine rota recombinant VP7 protein antigen antigen.
Step 2 middle and upper reaches primer P1 is 5 '-AGGCCATGGATGCACAAAACTACGGTAT-3 ', and downstream primer P2 is 5 '-CGCAAGCTTTTATTCCCTTGGCCCCAAT-3 '.
Along with the exploitation of the modern techniquies such as modern molecular immunology and molecular biology, for new way has been opened up in the development of biotechnology diagnostic reagent of new generation.The diagnostic antigen or the antibody that with the advanced technique means such as genetically engineered, cell engineering, obtain, its outstanding feature is that homogeneity is good, purity is high, high specificity, stable performance, and be easy to preserve and batch production, set up diagnostic techniques thus and method is easy to International standardization, be the main direction of following disease diagnosis development.Meanwhile, the diagnostic antigen of producing with biotechnology, can avoid loaded down with trivial details cell cultures to prepare a large amount of antigenic substances.
BRVVP7 albumen is comprised of 326 amino acid, molecular weight is 37kDa, account for 30% of rotavirus protein total amount, being in virus structural protein content more than second, is viral outer capsid albumen, and VP7 is in rotavirus main and antigen, determine its G serotype, therefore externally can induce virucidin, can excite stronger immunne response, the cloning of capsid protein VP7 gene, expression and expression product purifying thereof.No matter the protein product of its purifying, be the diagnostic antigen as a kind of disease,, as a kind of immunological reagent of excitating organism immunne response, is still all extremely useful novel biological agents.The further application of this preparation, will provide important basic substance to the diagnosis and treatment of BRV.
Restructuring BRVVP7 albumen prepared by the present invention has the immunobiology technical characteristic close with natural VP7 albumen; and can large-scale production; the method avoids taking the production method of vitro culture virus antigen, differential centrifugation and density gradient centrifugation purified virus; improve working efficiency, reduced nonspecific reaction.The antigen of producing with this, can avoid doing antigen with live virus and disseminate viral danger.
The bovine rota VP7 albumen of prokaryotic expression of purifying of take is envelope antigen, set up the indirect ELISA diagnostic method that detects bovine rota antibody, utilize indirect ELISA method and the virus neutralization tests of setting up to detect bovine serum, the coincidence rate of VP7 protein ELISA method and virus neutralization tests reaches 87.2% simultaneously.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of pcr amplification result in embodiment one step 2; Fig. 2 carries out for embodiment one step 2 is obtained to recombinant plasmid pMD18-T-VP7 the result figure that enzyme is cut evaluation; Fig. 3 is the SDS-PAGE electrophorogram of the recombinant protein after embodiment one step 5 purifying; Fig. 4 is the Western Blot qualification result of embodiment one recombinant protein.
Embodiment
Embodiment one: the preparation method of present embodiment bovine rota recombinant VP7 protein antigen antigen, carries out according to the following steps:
One, the extraction of bovine rota BRV-DQ pnca gene group RNA:
Bovine rota BRV-DQ strain is after the cracking of Trizol, the Virahol extracting that adds chloroform and 2 times of volumes of 2 times of volumes, the dehydrated alcohol precipitate nucleic acids that adds 2.5 times, the ice washing with alcohol that is 70% with concentration of volume percent again precipitation, after drying at room temperature, add appropriate aseptic deionized water dissolution precipitation, obtain BRV-DQ pnca gene group RNA ,-20 ℃ save backup.
Described bovine rota BRV-DQ strain is DQ-75, at separation and the genotype identification > > (Xie Jinxin etc. thereof of article < < new born bovine rotavirus, Heilongjiang Bayi Agricultural Reclamation University's journal, in October, 2010, the 22nd the 5th phase of volume) open in, can be obtained by author place.
Two, RT-PCR amplification VP7 gene:
The RNA reverse transcription that step 1 is obtained is cDNA, and concrete steps are carried out (described ThermoScript II is bought from Promega company) by the specification sheets of ThermoScript II.The nucleotide sequence of the cDNA obtaining, as shown in SEQIDNO:1, is used upstream primer P1 and downstream primer P2 to carry out pcr amplification to cDNA, according to the specification sheets of TaqDNA polysaccharase, carries out (described TaqDNA polysaccharase is bought from Fermentas company).The PCR product obtaining is connected with pMD18-T carrier after glue reclaims purifying, obtains recombinant plasmid pMD18-T-VP7.
Step 2 middle and upper reaches primer P1 is 5 '-AGGCCATGGATGCACAAAACTACGGTAT-3 ', and downstream primer P2 is 5 '-CGCAAGCTTTTATTCCCTTGGCCCCAAT-3 '.
Three, pMD18-T-VP7 plasmid and expression vector pET30a are all adopted to NcoI and HindIII double digestion, then by T4DNA ligase enzyme, connect, ligation is undertaken by the specification sheets of T4DNA ligase enzyme, connect product and proceed to Host Strains BL21, the correct bacterium of checking order is positive recombinant bacterium pET30a-VP7/BL21.
Four, protein induced expression:
The bacterium liquid of 50 μ L recombinant bacterium pET30a-VP7/BL21 is inoculated in to 5mL containing in the LB liquid nutrient medium of Kan+, and 37 ℃ of shaking culture are spent the night.Then the bacterium liquid of getting 100 μ L incubated overnight is inoculated in 10mL containing in the LB liquid nutrient medium of Kan+, and take 200rpm shaking culture to OD600 value for 37 ℃ is 0.6～0.7 o'clock, and sampling 1mL contrasts before as induction.Then, add IPTG to final concentration 1mmol/L, continue with 200rpm shaking culture, respectively at 2h, 3h, 4h, 5h and 6h sampling 1mL.In the centrifugal 1min of 12000rpm, collect bacterial sediment, add 50 μ L0.01mol/LPBS, add the resuspended precipitation of equivalent 2 * SDS sample loading buffer, 100 ℃ are boiled 5min, and the SDS-polyacrylamide gel electrophoresis with 12% detects expression.
Five, the purifying of recombinant protein:
(1) in the bacterium liquid of group bacterium pET30a-VP7/BL21, add IPTG to final concentration 1mmol/L, with 200rpm shaking culture, when the OD600 of bacterium liquid value reaches 0.6, the centrifugal 10min of 8000rpm collects thalline;
(2) supernatant discarded, every gram of wet bacterium adds the deionized water of 3mL sterilizing, adds proteinase inhibitor (buying from Amresco company) and the 80 μ L N,O-Diacetylmuramidases of 3 μ L50mmol/L after suspension mixes, and in room temperature, at horizontal shaking table, slowly shakes 15min.
(3) use ultrasonic disruption thalline, power 400W, broken 6s, interval 6s, repeats 100 times, then with the centrifugal 10min of 12000rpm, collecting precipitation.
(4) in precipitation, add 1mL sterilizing deionized water and equal-volume 2 * SDS sample loading buffer, boil 5min.
(5) the centrifugal 10min of 12000rpm, gets the whole loadings of supernatant, does discontinuous vertical electrophoresis, and voltage is 100V at first, when dyestuff is raised to 200V to voltage after separation gel, until dyestuff is run out of separation gel.
(6) take off the KCl solution 5min that gel is placed in ice-cold 0.25M, after gel band colour developing, with knife blade, cut the gel that contains object band.
(7) gel cutting is put into dialysis tubing, add 3mLTris-glycine electrophoretic buffer, 80V horizontal strip electrophoresis 2h, and then by dialysis tubing counterelectrophoresis 2min.
(8) use 0.01mol/LPBS damping fluid, to the albumen of wash-out in 4 ℃ of dialysis, is used PEG20000 protein concentrate, with gene quantification instrument, measures its concentration, gets 20 μ L albumen and carries out SDS-PAGE electrophoresis, and all the other are frozen standby in-70 ℃.
Six, the WesternBlot of recombinant protein identifies:
First carry out after SDS-PAGE electrophoresis, then use half dry type electricity transfer printing instrument to carry out protein immunoblotting reaction, protein delivery is carried out to Westernblot analysis to NC film, its concrete steps are as follows:
(1) transferring film: thing under placing successively on the negative plate of half dry type electricity transfer printing instrument: soaked transfer printing damping fluid 3 of filter paper, gel, nitrocellulose filter, soaked 3 of the filter paper of transfer printing damping fluid, drain as much as possible bubble, cover anode cover, constant voltage 9V, energising 1h
(2) sealing: NC film is put into plate, add confining liquid according to filter membrane area with 0.1mL/cm2 amount, drain bubble, then airtight plate slowly shakes on horizontal shaking table, room temperature sealing 2h or 4 ℃ spend the night.
(3) washing: PBST washes 3 times on shaking table, each 10min.
(4) combination of antibody: with confining liquid by primary antibodie by suitable proportion dilution, the film having sealed is placed in to antibody-solutions, drain bubble, room temperature is shaken 2h or 4 ℃ and is spent the night.
(5) washing: PBST washes 3 times on shaking table, each 10min.
(6) two anti-combinations: with confining liquid, the anti-ox IgG of the rabbit of HRP mark is diluted by a certain percentage, the film having sealed is put in antibody-solutions, room temperature is shaken 1h.
(7) washing: PBST washes 3 times on shaking table, each 10min.
(8) colour developing: develop the color after adding DAB.
(9) termination reaction: take out of now when NC film has color bar, the 5～10min that fully develops the color, is transferred to color development stopping reaction in deionized water by film, in order to avoid background color is too high.
(10) film is put on filter paper and is dried, analytical results.
In step 2, as shown in Figure 1, wherein M is DNA standard DL2000 to the electrophorogram of pcr amplification result, and 1,2 is expression product.
Step 2 is obtained to recombinant plasmid pMD18-T-VP7 and carry out enzyme and cut evaluation, as shown in Figure 2, wherein M is DNA standard DL2000, and 1 cuts product for enzyme.
As shown in Figure 3, wherein M is protein molecule quality standard to the SDS-PAGE electrophoresis of the recombinant protein after step 5 purifying, and 1 is the pET-30a+VP7 before induction, the 2 pET-30a empty carriers for induction 5h, 3 pET-30a+VP7 for induction 5h.
As shown in Figure 4, wherein M is protein molecule quality standard to the WesternBlot qualification result of recombinant protein, 1 contrast of the empty carrier for induction; 2 is induction after product.
Above the results show the inventive method has obtained bovine rota recombinant VP7 protein antigen antigen.
The bovine rota VP7 albumen of prokaryotic expression of purifying of take is envelope antigen, set up the indirect ELISA diagnostic method that detects bovine rota antibody, utilize indirect ELISA method and the virus neutralization tests of setting up to detect 78 parts of bovine serums simultaneously, detected result is in Table 1, the serum ELISA positive of examining is 47 parts, negative 31 parts; Positive 41 parts of neutralization test, negative 37 parts.The coincidence rate of VP7 protein ELISA method and virus neutralization tests reaches 87.2%.
Table 1
Claims (3)
1. bovine rota recombinant VP7 protein antigen antigen, is characterized in that this bovine rota recombinant VP7 protein antigen antigen is by 644bp alkali yl coding, and size is 40kDa, and base sequence is as shown in SEQ ID NO:1.
2. the preparation method of bovine rota recombinant VP7 protein antigen antigen claimed in claim 1, is characterized in that the method carries out according to the following steps:
One, extract the geneome RNA of bovine rota BRV-DQ strain; Two, RNA reverse transcription step 1 being obtained is cDNA, uses upstream primer P1 and downstream primer P2 to carry out pcr amplification to cDNA, and PCR product is connected with pMD18-T carrier after glue reclaims purifying, obtains pMD18-T-VP7 plasmid; Three, pMD18-T-VP7 plasmid and expression vector pET30a are all adopted to Nco I and Hind III double digestion, then by T4DNA ligase enzyme, connect, connect product and proceed to Host Strains BL21, obtain recombinant bacterium pET30a-VP7/BL21; Four, the abduction delivering of recombinant protein; Five, the purifying of recombinant protein, obtains bovine rota recombinant VP7 protein antigen antigen.
3. the preparation method of bovine rota recombinant VP7 protein antigen antigen according to claim 2, it is characterized in that step 2 middle and upper reaches primer P1 is 5 '-AGGCCATGGATGCACAAAACTACGGTAT-3 ', downstream primer P2 is 5 '-CGCAAGCTTTTATTCCCTTGGCCCCAAT-3 '.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110903403A (en) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | Bovine rotavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibody |
| CN114478714A (en) * | 2022-02-04 | 2022-05-13 | 通化师范学院 | Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein |
-
2014
- 2014-09-11 CN CN201410461064.1A patent/CN104211786A/en active Pending
Non-Patent Citations (4)
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| XIE J等: "GU144587", 《GENBANK》 * |
| 张冠群: "表达牛轮状病毒VP7蛋白重组干酪乳杆菌的构建及其免疫学初步分析", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 杨少华等: "A组牛轮状病毒基因工程亚单位疫苗的初步研究", 《中国人兽共患病学报》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110903403A (en) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | Bovine rotavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibody |
| CN114478714A (en) * | 2022-02-04 | 2022-05-13 | 通化师范学院 | Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein |
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