Establishment Of Indirect ELISA Based On Recombinant NS4 Protein For The Detection Of Antibodies Against Bluetongue Virus

This paper mainly takes Bluetongue virus(BTV)as the experimental research object,carries on the construction of its NS4 gene prokaryotic expression vector,and achieves the large number of high efficient expression of NS4 protein.And then further prepares the BTV NS4 prokaryotic expression recombinant protein polyclonal antibody.Finally,the purified fusion protein p ET-32a-NS4 was used as the coating antigen,and the optimal coating amount,the optimal serum dilution,the optimal working concentration and the critical value of reaction were screened.The BTV indirect ELISA method based on NS4 protein was established,and antibodies were detected and analyzed in 56 bovine clinical serum samples The main contents and results of this study are as follows:(1)Construction and expression of BTV NS4 gene prokaryotic expression vectorIn this study,The NS4 gene was amplified by PCR and connected with empty vector p ET-32a-NS4 after double enzyme digestion to construct p ET-32a-NS4 prokaryotic expression vector.p ET-32a-NS4 was transformed into E.coli BL21 cells and expressed by IPTG induction.The results showed that the prokaryotic expression vector p ET-32a-NS4 was successfully constructed.The optimal IPTG induction concentration was 0.8mmol/L for 6h,and the recombinant protein was mainly expressed as inclusion body.Western blot showed that the target band was visible at 27 k Da,which was consistent with the expectation.p ET-32a-NS4 prokaryotic expression vector was successfully constructed to achieve a large number of NS4 protein and efficient expression,which laid a foundation for further preparation of antibodies against NS4 protein.(2)Preparation of polyclonal antibody against BTV NS4 prokaryotic expression recombinant proteinPolyclonal antibodies were prepared by immunizing New Zealand white rabbits with the purified fusion protein p ET-32a-NS4.The results showed that the titer of the antibody was over 1:52000,the purity was over 85%,and the yield of the purified antibody was over 8.0mg.The recombinant protein p ET-32a-NS4 was used to prepare the antigen and inoculated New Zealand white rabbits.Polyclonal antibodies with good immunogenicity were obtained,which laid a foundation for the detection of BTV NS4 protein and the study of indirect ELISA method.(3)Establishment of indirect ELISA method for detecting BTV NS4 proteinThe purified fusion protein p ET-32a-NS4 was used as the coated antigen,and the optimum coating amount,serum dilution,Optimum sealing fluid,working concentration of enzyme-labeled antibody and critical reaction value were selected.The BTV indirect ELISA method based on NS4 protein was established,and antibody detection was analyzed in 56 bovine clinical serum samples.The results showed that the positive coincidence rate of antibody was 100%,and the indirect ELISA method based on NS4 had good sensitivity and repeatability.