Preparation Of Monoclonal Antibodies And Screening B Cell Epitopes For Bluetongue Virus Type 1 And 16 VP2 Protein

Bluetongue(BT)is a non-contact and violent infectious disease caused by the Bluetongue virus(BTV),which is mainly transmitted by arthropods.There are many serotypes of BTV,and there is no cross-protection between the serotypes.The VP2 protein is located in the outermost layer of virus particles,which can stimulate the body to produce a protective immune response and is the main antigenic determinant.Currently,a total of 29 BTV serotypes have been identified,of which BTV1 and BTV16 are the main epidemic strains in China.Early diagnosis is very important to control the spread of the epidemic.Monoclonal antibodies with good specificity and stability can be used in diagnostic technology and precise prevention and control of epidemics.Therefore,the preparation of monoclonal antibodies against BTV1 VP2 and BTV 16 VP2 protein in this study and their epitope identification are of great significance for the diagnosis and prevention of BTV 1 and BTV 16 and the research of new vaccines.Primers were designed according to the BTV1 L2 and BTV16 L2 gene sequences included in Genbank,and the VP2 target genes of BTV1 and BTV 16 were used as templates to amplify the 63～471 gene fragment of 63～471 amino acid,and inserted into the pGEX-6p-1 vector to construct Recombinant expression plasmids pGEX-6p-1-1tVP2 and pGEX-6p-1-16-tVP2.The recombinant plasmid was transformed into BL21(DE3)competent cells,and the expression was induced and then identified,and the protein was purified by GST affinity chromatography.Western-blot results showed that BTV1 tVP2 and BTV 16 tVP2 can react with BTV1 and BTV16 positive sera,respectively,indicating that recombinant BTV1 tVP2 and BTV 16 tVP2 proteins have good immunoreactivity.The purified BTV1 tVP2 and BTV16 tVP2 were mixed as immunogens to immunize Balb/c mice,and monoclonal antibodies were prepared using hybridoma technology.Five hybridoma cells that stably secrete monoclonal antibodies against BTV1 and BTV16 VP2 proteins were screened out.They are named 4D12,8F2,5C9,10E9 and 10D12.Indirect immunofluorescence assay(IFA)results of cross-reactivity identification showed that monoclonal antibodies 8F2,10E9 and 10D12 can specifically react with eukaryotically expressed BTV1 tVP2 and BTV16 tVP2.Three monoclonal antibodies were used to prepare ascites by in vivo induction method,and the titer of ascites after purification was between 1:1.28×105～1:5.12×105.According to the overlapping peptide method,the amino acid sequence of the BTV1 tVP2 protein was designed,and 17 short peptides(L1～L17)were synthesized,each overlapping 10 amino acid residues,to identify the linear B cell epitope region recognized by the monoclonal antibody.Enzyme-linked immunosorbent assays(ELISA)and Dot enzyme-linked immunosorbent assay(Dot-ELISA)results show that 8F2,10E9,and 10D12 jointly recognize L15(401-435aa).L15 was further truncated(L15-1～L15-4),and the truncated synthetic peptides were screened and identified,and a new linear B cell epitope of BTV1 VP2 protein was screened out.The epitope sequence is:40IREQEKYIYGRVNLFD415.In this study,five monoclonal antibodies against BTV1 VP2 and BTV16 VP2 were prepared,and a new B cell epitope was screened and identified,which provided a basis for the precise prevention and control of BT,and laid a foundation for serological detection of BT and development of novel peptide vaccine.