Study On Indirect ELISA To Detect Antibody Against EMCV With Recombinant VP1 Epitope Protein

Encephalomyocarditis virus (EMCV) is a new kind of zoonoses virus, which has not been found in China before. EMCV is a member of Picornaviridae. Rodents are the natural hosts of EMCV, in which the virus usually persists without causing disease. The virus is transmitted from rodents to a wide variety of animal species and is not limited to a defined geographical location. EMCV has been recognized as a pathogen of pigs for many years. However, the clinical signs of disease are different, including sudden death of piglets and reproductive failure in sows. In general, Picornaviridae express four different capsid proteins, VP1, VP2, VP3 and VP4, among which VP1 is the most antigenic.In this report, we established an indirect ELISA to detect the antibodies against EMCV with recombinant VP1 epitope protein in porcine serum, and found that EMCV exists in China.1. Expression and antigenicity of the epitope of VP1 gene of EMCV in E. coliA 150-base-pair (bp) double strands DNA of the epitope of the VP1 coding gene obtained from genbank of EMCV was synthesized artificially, which had Bgl II and EcoR I restriction enzymes site on the 5' ends and 3' ends, respectively. And it was inserted into plasmid pGEX-6p-1 to construct the recombinant prokaryotic expression vector. After transformating the recombinant plasmid into E.coli competent cells BL21(DE3), the fusion protein was expressd by isopropyl- β -D-thiogalactoside (IPTG) ,and purified through GSTrap FF column. It was visualized on 12% of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with relative molecular weigh of 30 800.To evaluate the antigencity of the recombinant protein, it was injected into two rabbits, and the serum antibody against the protein was made and estimated with indirect ELISA and Western-blot.