Cloning And Sequence Analysis Of FMDV Structural Gene And Expression Of VP1, Development Of Indirect ELISA

FMD is a virulent infectious disease caused by FMDV that infects artiodactyl such as cattle, sheep, pig, et al. Outbreak and prevalence of the disease endanger the good development of graziery seriously and cause great financial loss, so all the world highlight study and contol of the disease. Capsid of FMDV is composed of VP1, VP2, VP3, VP4 of 60 copies, of which VP1 protein is called 1D protein, too. Its gene is in 2977-3615 of genome and code for 213 amino acids. VP1 is located on the surface of virus and is the main component that induces neutralizing antibody.Material of the study was FMDV from cell culture, nucleotide sequence of structural gene of FMDV and FMD vaccine strain were acquired by RT-PCR. Compared with FMDV reference strains, it was found that there was difference in VP1 gene that induces neutralizing antibody, and Phylogenetic tree was drawn according to VP1 gene sequence(VP1 gene sequence is the criterion of drawing Phylogenetic tree in the world). Compared with FMD vaccine, it was found that there was difference in VP2, VP3, not VP1.VP1 target gene was amplified by PCR with plasmid pMD18-T-VP1 as template, then it was cloned to vector pMD18-T and transform TG1 strain of E.coli, so recombinant plasmid pMD18-T-VP1 was acquired, sequencing assured that the sequence of VP1 was correct. Recombinant plasmid pMD18-T-VP1 and prokaryotic expression vector pET-30a(+) were cut by the same restriction endonucleases SacI and HindIII and ligated together, transformed TG1 strain of E.coli with ligation mixture, then the positive clone pET-30a(+)-VP1 was identified. Transforming BL21(DE3)pLysS with positive plasmid pET-30a(+)-VP1, inducing expression of VP1 gene with IPTG, collecting bacteria at different times, then doing SDS-PAGE, Western blotting and dot-ELISA analysis, the result showed that the gene of VP1 had been expressed well in E.coli and molecular weight of the target protein was 34ku, but the product did not have antigenicity.Recombinant plasmid pMD18-T-VP1 and eukaryotic expression vector pBlueBacHis2A were cut by the same restriction endonucleases SacI and HindIII and ligated together, transformed TG1 strain of E.coli with ligation mixture, then the positive clone pBlueBacHis2A-VP1 was identified. Cotransfecting insect cell Sf9 with plasmid pBlueBacHis2A-VP1 and Bac-N-Blue^TM DNA, screening recombinant virus by plaque, Sf9 cells were inoculated by pure recombinant virus, then 32.6 ku target protein was expressed, dot-ELISA analysis showed that the product had antigenicity.Indirect ELISA that can detected the valence of antibody was developed with the fusion protein expressed by eukaryotic expression system as antigen.