Expression And Of Bluetongue Virus Type 1 VP5 Protein And Preparation Of Monoclonal Antibodies

Bluetongue disease(BT)is a non-contact infectious disease caused by bluetongue virus(BTV),which is transmitted among a variety of ruminants by vector such as Cumulus.Infected animals usually have symptoms such as fever,cyanosis of the tongue,mucosal erosion,and tissue necrosis.BTV genome is composed of 10 double-stranded RNA.The 10 dsRNA encoded 7 kinds of the structural protein and 5 kinds of the non-structural protein respectively.The VP5 protein is one of BT serotype specific antigens,which is encoded by the M5 gene and constitutes the outer shell of BTV together with VP2 protein.In this experiment,BTV1 VP5 protein was successfully induced with optimal conditions,the medium is 2×YT medium,the concentration of IPTG is 0.4 mM,and the tempreture is 25?.And the VP5 protein can react specifically with anti-BTV1 positive serum.The purity of the protein is up to 85%,the concentration of the protein is 0.5 mg/mL.The serum titer of immunized mice measured by indirect ELISA method is 1:1.024×105.After cell fusion,16 hybridoma cell lines stably secreting anti-BTV1 VP5 monoclonal antibodies were prepared.The IPMA experiment showed that 8 of 16 cells specifically bound to VP5 eukaryotic protein,namely 1A6,1E9,4B9,5F11,6H11,8B4,12G5,12G10.VP5 protein is divided into 8 segments according to the results of bioinformatics analysis,each segment contains 75 amino acids,and 10 amino acids overlap in adjacent segments.8 truncated protein expression strains of VP5 protein were constructed,7 truncated proteins P-2-P-8 were successfully expressed in LB medium,0.1 mM IPTG and 16?.The purity of truncated protein is up to 95%,and the highest concentration of the 7-segment truncated protein is 1 mg/mL.Indirect ELISA showed that 1A6,1E9,4B9,5F11,6H11,8B4 cells can recognize P-3(130-205 aa)segment,12G5 can recognize both P-3(130-205 aa)and P-4(195-270 aa)segment,and 12G10 can recognize P-4(195-270 aa)segment.P-3 and P-4 were further truncated and synthesize polypeptides.Peptide-ELISA and Dot-ELISA experiments showed that 8B4 can recognize the T-2 segment,which can recognize the 151-180 aa segment of the VP5 protein.In the study,the monoclonal antibodies were prepared and the recognition region of the mAbs were identificated.The result of this reasearch is useful for the screening of the VP5 protein epitope.And it is of great significance to lay the foundation for establishment of BTV1 type specific test methods and reaserch about VP5 function.