Cloning Of The Structural Protein VP2-3-1 Gene Of Foot-and-Mouth Disease Virus And Its Prokaryotic Expression

Foot-and-Mouth Disease (FMD) is a severe disease, caused by foot-and-mouth disease virus, of cloven-hoofed animals, particularly swine, cattle and sheep. FMD seriously impair healthy development of animal husbandry and result in considerable economic losses.Therefore, all the countries of the world pay much attention to its study and prevention.In this paper, FMDV PI gene was amplified from positive plasmid AHP1 with a pair of primers which was designed according to sequence of FMDV China-99 strain. PCR product of P1 gene was digested with NcoI and XhoI, and got interest gene VP2-3-1.The interest gene was ligated into vector pProEX-HTb which was digested with Ncol and Xhol respectively, then transformed into E.coli BL21(DE3) cells. Recombinant plasmid were identified by restriction analysising and PCR and DNA sequencing. A recombinant plasmid with cDNA of FMDV VP2-3-1 gene was selected ,named as pProEX-VP2-3-1.The bacteria containing pProEX-VP2-3-l was induced with Isopropyl-D-galactoside(IPTG) and samples of bacteria culture fluid were collected at different time respectively. The samples were examined by SDS-PAGE and Western blotting after being properly treated.The results showed that the interest gene was successfully expressed in E.coli and can be recognized by the positive bovine serum of FMDV. The amount of target protein occupied 18%~25% of the total protein in bacteria lysate. It was proved that the expressed protein was insoluble and existed in the form of inclusion body .Target protein with some activities was gotten by denaturing the inclusion body, purifying and refolding expression protein.These results will be contributed to establish a new diagnosis method with genetically expressing protein.