Study On The Antigenicity Of CSBV Recombinant Structural Protein And Development Of Double Sandwich ELISA Kit

Chinese Sacbrood Disease(CSBD)is a highly pathogenic and infectious viral Disease caused by Chinese Sacbrood Virus(CSBV).However,there is no suitable bee virus culture system including CSBV,which greatly restricts the research and prevention of CSBV.On the detection of CSBV,symptom diagnosis is not exact,electron microscope and serological detection a laborious,especially difficult to obtain CSBV standard positive serum,made by immunological methods for detecting CSBV rarely make the news research,while molecular biology diagnosis is faced with the problem of high cost,need special laboratory equipment,can only be used in laboratory tests,not large area promotion.In view of this,this research in our laboratory CSBV isolated gene as the foundation,respectively,to optimize the modified with would connect VP1,VP2 gene and the optimized continuous encoding VP2,VP4 and VP1 gene of target genes,period of prokaryotic expression,expression and purification of proteins immune mice and chickens,compare these two expression protein antigenicity,select antigenicity good protein instead of CSBV as immunogen,preparation of monoclonal antibodies.Double antibody sandwich ELISA kit was prepared by using monoclonal antibody,and the performance of the kit was tested.The specific research content is as follows:1.Antigenicity comparison of target proteins.Opti VP1 and Opti VP2 were synthesized by optimization and codon modification based on the gene sequence of CSBV isolated in our laboratory(Genbank:KU574661.1).The target genes VP2-P-VP1 and VP2-VP4-VP1 were obtained by fusion PCR using rigid peptide as Linker and optimized VP4 gene.They were constructed into p ET28 a vector and induced expression.Mice were immunized with CSBV,PBS and purified proteins.Serum titer and lymphocyte proliferation index were detected.It was found that the VP2-P-VP1 group was slightly higher than the VP2-VP4-VP1 group,with no significant difference(P>0.05).Both groups were significantly higher than th e PBS group,but lower than the CSBV group,with significant difference from the CSBV group(P<0.05).The Ig Y titer was detected by immunizing chickens with these two proteins,and it was found that Ig Y could be induced in both groups,which was lower than that in CSBV group,and Ig Y titer in VP2-P-VP1 group was slightly higher than that in VP2-VP4-VP1 group.Through virus neutralization test,Ig Y produced by these two groups was found to have virus neutralization ability.It indicates that VP2-P-VP1 and VP2-VP4-VP1 proteins have good immunogenicity,among which VP2-P-VP1 has slightly higher antigenicity and can be used as an immunogen instead of CSBV for the research and development of biological agents.2.Preparation of monoclonal antibody against CSBV.Monoclonal antibodies were prepared by immunizing mice with purified VP2-P-VP1 protein.A total of 8 positive cell lines were screened,including 1E2,2E7,3B12,4B1,4G1,4D4,4F4 and 5D5.The antibody titer was stable after 10 successive passages.The 8 strains of cells were injected into the abdominal cavity of mice to prepare ascites,and the titers reached more than 16,000,among which the titers of 4F4 and 5D5 reached 64,000.Subtypes identified: 2E7,3B12,4B1 and 4D4 are Ig G1,4G1 and 4F4 are Ig G2 a,1E2 and 5D5 are Ig G2 b.After purification of ascites and SDS-PAGE analysis,it can be seen that the heavy chain band of 55 k D and the light chain band of 25 k D are obviously visible,and there are few heteroproteins,so the purification effect is better.VP1 purified protein and VP2 purified protein were coated by ELISA,and the binding sites of 1E2,2E7,3B12,4B1 and 5D5 were on VP1,while the binding sites of 4G1,4D4 and 4F4 were on VP2.ELISA detected the specificity of monoclonal antibodies and found that MAb only reacted with CSBV and not with other viruses,indicating that the prepared monoclonal antibodies had good specificity.Neutralization test showed no significant difference in mortality between 4F4 group and 5D5 group and PBS group(P>0.05),and significant difference with CSBV group(P<0.05).The mortality of the other 6 groups was not significantly different from that of CSBV group(P> 0.05),indicating that 4F4 and 5D5 have neutralization effect on CSBV and can be used for the prevention and t reatment of CSBV.3.Development of double-sandwich ELISA.It was found that 1E2 and 5D5 had the best pairing effect.The chessboard method was used to determine the optimal coating concentration of 1E2 was 3.75 μg /m L,and the optimal dilution concentration of HRP labeled 5D5 as 1:2000.Then,the coating time,sealing solution and sealing time,antigen incubation time,detection antibody incubation time,color development time and other reaction conditions were o ptimized,and a double-antibody sandwich ELISA method was established.The specificity experiment showed that it only reacted with CSBV,and did not cross-react with other bee viruses,so it had good specificity.The minimum detection quantity of CSBV virus was 3.675 × 104 copies/u L.The variation coefficients of both inter-batch and intra-batch repeated tests were less than 5%,indicating that the method had good repeatability.Sixty bee larvae were tested by double sandwich ELISA and verified by RT-PCR.The results showed that the positive coincidence rate of double sandwich ELISA and RT-PCR was 88.9%,the negative coincidence rate was 91.7%,and the total coincidence rate was 90%,indicating that the double-sandwich ELISA method established has good detection effect and can be used for clinical sample detection.According to the "verification principle of infectious disease diagnostic test" recommended by OIE,the kit was assembled,and after the stability test,the shelf life of the kit was 12 months.In conclusion,VP2-P-VP1 and VP2-VP4-VP1 proteins were successfully expressed in this experiment.Through comparison,8 strains of monoclonal antibodies were prepared by selecting VP2-P-VP1 proteins with good antigenicity.Two of the 8 strains have the ability of virus neutralization and can be used in the development of anti-csbv biological agents.A double-sandwich ELISA kit with high specificity,sensitivity and accuracy has been successfully developed,which can be used for clinical detection of CSBV,provi ding technical support for rapid diagnosis,real-time monitoring and early warning of CSBV.