Fusion Expression Of Bluetongue Virus Type 1 And 16 VP2 Protein And Preparation Of Monoclonal Antibody

Bluetongue virus(BTV)is the causative agent of Bluetongue(BT),which can cause hemorrhagic disease in ruminants.The viral outer capsid consists of VP2 and VP5,which interact with VP5 to promote viral attachment and entry;the inner capsid can be divided into two concentric protein layers that surround the viral core complex(RNA genome and minor proteins VP1,VP4 and VP6).The outer layer consists of260 trimers of VP7 protein molecules arranged in icosahedrons,and the inner layer consists of VP3 protein pentamers.The outer capsid protein VP2 is located in the outermost layer of the BTV particle structure.This protein can induce the body to produce antibodies and determine the serotype of BTV.Early diagnosis and vaccination are of great significance in controlling the spread of the disease.The expression of the fusion protein and the development of monoclonal antibodies in this study laid the foundation for the diagnosis of BTV1 and BTV16 and the research of new vaccines.The gene sequences of the well-conserved and immunogenic regions(amino acid residues 63～471)in BTV1 VP2 and BTV16 VP2 were intercepted,and overlap extension PCR was used.The gene sequences of BTV1 t VP2 and BTV16 t VP2 were fused,and the fusion gene was cloned into the p GEX-6p-1 vector to construct a recombinant expression vector p GEX-6p-1-tf VP2 that truncated the fusion VP2protein(tf VP2).The tf VP2 protein was expressed in the prokaryotic expression system,and the soluble expression effect of tf VP2 protein was the best when the concentration of inducer(IPTG)was 0.2 mmol/L,and the expression was induced at26℃for 12 h.The tf VP2 protein was purified by GST affinity chromatography to a purity of 90%.Indirect enzyme-linked immunosorbent assays(ELISA)were used to compare the immunization effect of immunization with tf VP2 protein as immunogen and mixed immunization with BTV1 t VP2 protein and BTV16 t VP2 protein as immunogens.The results showed that immunized mice with tf VP2 protein as immunogen,the serum titer of mice could reach 1:2.048×10~5,which was higher than that of mixed immunization group(1:2.56×10~4～1:5.12×10~4).Four hybridoma cell lines that stably secrete anti-tf VP2 protein、BTV1 VP2protein and BTV16 VP2 protein monoclonal antibodies were obtained by cell fusion technology and limiting dilution,named 1G8,1G10,2C2 and 6E4,respectively.Indirect immunofluorescence assay(IFA)was used to detect the reactivity of hybridoma cell lines with recombinant proteins expressed in eukaryotes,the results showed that all four hybridoma cell lines could react with eukaryotic expression of tf VP2 protein,BTV1 t VP2 protein and BTV16 t VP2 protein.Detection of the reactivity of hybridoma cell lines with inactivated virus by Dot enzyme-linked immunosorbent assay(Dot-ELISA),the results showed that the four hybridoma cell lines could react with BTV1 inactivated virus and BTV16 inactivated virus.Three hybridoma cell lines with higher titers,1G10,2C2 and 6E4,were selected,and ascites was prepared by in vivo induction method.The titers of the purified m Abs 1G10,2C2and 6E4 were 1:5.12×10~5,1:2.56×10~5 and 1:5.12×10~5,respectively,and the affinity constants were 8×10~9 L/mol and 1.9×10~9 L/mol,8.6×10~9 L/mol,respectively.The BTV1 t VP2 protein was truncated and expressed in segments(N1～N4).Indirect ELISA and Dot-ELISA experiments showed that the m Abs could recognize N3(243～382 aa)and N4(333～471 aa)at the same time.The synthetic peptides L1and L2 were designed,and the reactivity of the monoclonal antibody with the peptide was detected by Dot-ELISA and Peptide-ELISA.The results showed that the four monoclonal antibodies could recognize the synthetic peptide L1(326～360 aa).Further truncation of L1(L1-1 and L1-2)found that 1G10,2C2 and 6E4 could recognize L1-2(338～360 aa),which could recognize 338～360 aa of BTV1 VP2protein.In this study,monoclonal antibodies against BTV1 VP2 protein and BTV16 VP2protein were prepared,and the epitope regions recognized by the monoclonal antibodies were identified.It laid the foundation for the research of new bluetongue vaccine,the establishment of detection method and the screening of BTV1 VP2protein epitope.