Expressing Of Epitope-chimeria Of Foot-and-mouth Disease Virus By Bacillus Subtilis

Foot-and-mouth disease（FMD) is one of the major infectious diseases which is a threat tohealthy development and stability of livestock. FMD mainly infects cloven-hoofed animals, such as pigs,cattle, sheep, camels, antelopes, deer, etc. FMD spreads fast and form large-scale epidemic in a shorttime, which has brought huge economic losses to the relevant country (region) and seriously restrict thedevelopment of animal husbandry. At present, China’s prevention and control strategies of FMD ismainly immunization, but the Inactivated whole virus vaccine commonly have a variety of deficienciesin used, The vaccines based on the development of genetic engineering technology has potentialapplications. In this study, the virus-like particles (VLPs) formed by the porcine circovirus type2capsidprotein show a neutralizing epitope of O type FMD virus, the main purpose is to develop epitope-basedvaccines for FMD.The capsid of FMD virus is an Icosahedron, which is composed by60asymmetric original tabletsmade up of the four structural protein VP1, VP2, VP3, VP4. The obvious feature of FMDV particlesurface is that a ring (GH loop) from the VP1push out from the surface, showing the mobility on thephysical and the versatility on the biochemical, G-H ring contain highly conserved arginine-glycine-aspartate (Arg-Gly-Asp)(RGD) motif which is both the main ingredients of cell adsorption sites andimportant neutralization site. The G-H loop spans140to160amino acid residues of VP1, while theother parts of the capsid have no significant changes, The G-H loop will adopt different conformations,showing flexibility.The capsid protein of Porcine circovirus type2(PCV2) was adopted as a protein skeleton, and itsneutralization epitope was replaced by the G-H loop of VP1of foot-and-mouth disease virus, strainO/Mya-98/2010, which was used to constitute a chimeric capsid protein designated as Cao.Subsequently, based on plasmid pHCMC05, an E.coli-Bacillus subtilis(Bs) shuttle plasmid p7257-P43was constructed by inserting of p43promoter (P43) and neutral protease B signal peptide (nprBsp) ofBs orderly into its multiple cloning site (MCS). Thereafter, the Cao gene was synthesized artificially, and was induced into MCS following the nprBsp to complete the secretary expression plasmidp7257-P43-Cao. The expressing plasmids were transformed into the bacillus subtilis strain WB800forinduction expression by chloromycetin（Cm）. SDS-PAGE electrophoresis showed that Cao could beexpressed by Bs WB800strain secretively, and Western blot demonstrated that the chimeric protein hadimmunoreactive activities. By electron microscopy observation, those chimeric capsid proteins can selfassembled to VLPs in vitro which diameter is about15±2nanometer.