Screening And Identification Of B Cell Epitopes Of Structural Protein Of Foot-and-Mouth Disease Virus Type A Strain A/WH/09

Foot-and-mouth disease (FMD) is an acute, highly contagious disease of cloven-hoofed animals caused by the foot-and-mouth disease virus (FMDV). Foot-and-mouth disease virus (FMDV) belongs to the aphthovirus genus of the Picornaviridae family and Seven distinct serotypes of FMDV have been defined, namely types O, A, C, Southern African Territories (SAT) 1, SAT2, SAT3 and Asia1,and each strotype has no cross immunity with others. B cell epitopes of FMDV are immune active regions of antigen molecules,which can be recognized by B cell receptor(BCR) and induce specific antibody. In our study, The B cell epitopes of structural protein of FMDV A/WH/09 were analyzed and identified by the means of bioinformatics in combination with molecular biology methods. The results would be particularly useful for further studying the structure and function of P1 protein and designing the epitope vaccine for foot-and-mouth disease (FMD) type A.In the present study, amplification primers were designed based on the P1 gene sequences of FMDV type A available in GenBank. P1 gene of strain A/WH/09 were amplified by RT-PCR from messenger RNA purified from neonate rat carcass infected by FMDV strain A/WH/09, and cloned into pGEM-T easy vector to construct the recombinant plasmid pGEM-T-P1. The result showed that the new gene has high similarity with other reference gene in GenBank in nucleotide and amino acid sequence. Several analysis method were used to predict B cell epitopes of P1,and corresponding epitope peptide fragments were synthesized.After then, prokaryotic expression primers were designed according to the sequence of the gene, and the pGEM-T-P1 recombinant plasmid was used as template in the PCR reaction to amplify the fragments encoding P1. The amplified products and prokaryotic expression vector pET-30a(+)were digested by restriction enzyme respectively,and the digested products of P1 were ligated to the pET-30a(+), then transformed into BL21(DE3) host cells. Expression of the recombinant plasmid was induced with IPTG, and resulted in a high level of protein expression. SDS-PAGE experiments revealed that molecular weight of P1 is about 80.754kDa. Soluble analysis shows that the expressed product was inclusion body.After inclusion body prepared and fusion protein purified, the purified product was emulsified with Freund's adjuvant,then New Zealand rabbits were immunized to prepare polyclonal antibody against P1.The ELISA titer of rabbit anti-FMDV-P1 serum was about 1:12800.Western blot analysis showed that the antiserum could bind to the expressed P1 protein specifically.Synthesized epitope peptide fragments, prepared polyclonal antibody and goat anti-rabbit IgG labeled by HRP were used as antigen, first antibody and second antibody respectively to screen and identify the B cell epitopes of P1 by indirect ELISA,the result revealed that the fragment 90～106aa and 130～148aa of VP1, 14～27aa and 126～144aa of VP3 showed excellent antigenicity. Screening and identification of main B cell epitopes of A/WH/09 lay foundation for further study on the multi-epitope vaccine of FMD type A.