Screening And Identification Of Main B Cell Epitopes Of Foot-and-mouth Disease Virus Type A Strain Af/72

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. 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 the present study, amplification primers were designed based on the gene sequences of FMDV type A available in GenBank. P1 and 2ABC gene of strain AF/72 were amplified by RT-PCR from messenger RNA purified from cryopreserved BHK-21 cell cultures infected experimentally with FMDV strain AF/72, and cloned into pGEM-T easy vector to construct the recombinant plasmid pGEM-T-P1, pGEM-T-2ABC for sequencing. The result showed that the new genes we obtained have high similarity with other reference gene in GenBank in nucleotide and amino acid sequence, with similarity above 92% and 94% respectively. Based on homology modeling of P1 and 2ABC,several analysis method were used to predict B cell epitopes of VP2,VP3,2C,and corresponding epitope peptide fragments were synthesized.After then, prokaryotic expression primers were designed according to the sequence of the genes, and the pGEM-T-P1 and pGEM-T-2ABC recombinant plasmid used as template in the PCR reaction to amplify the fragments encoding VP2,VP3,2C. The amplified products and prokaryotic expression vector pET-28a(+)were digested by restriction enzyme respectively,and the digested products of VP2,VP3,2C were ligated to the pET-28a(+), 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 VP2 is about 25KDa,VP3is about 24KDa,, and 36KDa for 2C. Most of the expressed products were inclusion body.After inclusion body prepared and fusion protein purified, the purified products were emulsified with Freund's adjuvant,then New Zealand rabbits were immunized to prepare polyclonal antibody against VP2,VP3,2C. ELISA and Western Blot demonstrated that the polyclonal antibody had obvious specificity and the titers were all above 1: 16000.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 VP2,VP3,2C by indirect ELISA,the result revealed that the fragment 1aa-12aa and 165aa-183aa of VP2,the fragment 30aa-46aa and 128aa-142aa and 206aa-221aa of VP3 and fragment 1aa-16aa and 41aa-56aa of 2C showed excellent antigenicity. Screening and identification of main B cell epitopes of AF/72 provided valuable technical parameters for further study on the multi-epitope vaccine of FMD type A.