Prokaryotic Expression Of Porcine Foot-and-mouth Disease Virus Type O VP₁ Gene And Detecting Of Re-actinogenicity Of The Recombinant Protein

Foot and mouth disease virus(FMDV) causes a highly contagious infectious disease foot-and-mouth(FMD) in cloven-hoofed animals. This disease is widely endemic all over the world and often results in considerable economic losses. FMDV capsid contains 60 copies of each of four structural proteins,VP₁,VP₂,VP₃ and VP₄.VP₁ protein is composed of 213 amino acids, also named 1D protein, occupying 2977-3615 of FMDV RNA genome. VP₁ plays a major role in inducing FMDV neutralization antibody.The study was aimed at porcine Foot and mouth virus (FMDV) type O TL-69 strain. According to the published VP₁ sequences, a pair of primers V₁/V₂ was designed and synthesized. A fragment of 600bp of VP₁ gene was amplified by RT-PCR and digested with BamH I /HindIII restriction endonucleases then ligated with pET30a+ vector to obtain a recombinant plasmid PETVP₁. The homology of the sequences of VP₁ gene among strain TL-69 and other strains of FMDVs registered in Genbank was compared to find that TL-69 , taiwan and chupei taiwan strains were highest and reached 98%. After the plasmid was transformed into E.coli host strain BL21(DE3), and tested by SDS-PAGE, the foreign protein was expressed efficiently in Ecoli when inducing with IPTG at 1.0 mmol/L for 4 hours. The expressed protein formed inclusion bodies in E.coli and the molecular mass was approximately 35kD as expected. After denaturation, purifycation and refolding, the purified soluble protein was examined by Western blot and indirect ELISA against the positive bovine serum specific to FMDV to show that the obtained recombinant protein has good re-actinogenicity. To establish basis for the further research and production of FMD genetic engineering vaccines and diagnostic kits.