Prokaryotic Expression Of The Structural Protein Of Foot-and-mouth Disease Type C And Immunogenic Analysis

Foot-and-mouth disease virus (FMDV) is the causative agent of a fulminating infectious disease of artiodactyls called Foot-and-mouth disease (FMD). This disease arouses worldwide concern because of its highly contagious and economically devastating destructive power. Although C type FMD has smaller infection range than the other six serotypes, it has already been prevalent in our surrounding countries. It is necessary for us to make more effort on the development of vaccine against serotype C.Currently the inactive vaccine is still the main biological product to prevent and control FMD, but it faces many challenges such as the security problem in production, transportation and usage. Therefore, it is necessary to develop novel FMD vaccines with more safety and efficiency. A reliable and popular alternative is the genetic engineering subunit vaccine. The main method to prepare immunogen is to exogenously express the structural proteins of FMDV. Escherichia coli (E. coli) expression system is characterized wih high expression level, easy product purification process and low cost. In the study, E. coli was chosen to express VP1, VP3, and VP0 structural proteins of the FMDV type C. pET-30a, pET-32a, and pGEX-6p-1 vectors were used to express those proteins. Different temperatures and inducer concentrations were explored to try to achieve the potential soluble expression. The results showed that genes encoding three structural proteins were expressed in the forms of inclusion bodies.Furthermore, the recombinant E.coli pET-30VP1, pET30VP3, and pET-30VP0 were collected after induction and the inclusion bodies in the cracked pellets were washed with 2M urea solution. SDS-PAGE showed a satisfactory washing effect with high purity. The inclusion bodies were dissolved with 8M urea solution after washing, and SDS-PAGE showed the three recombinant proteins were well dissolved in high concentration denaturant. The fusion proteins pET-30VP1 and pET-30VP3 successfμLly refolded after dialysis against low concentration urea. The renatured proteins could strongly react with anti-FMD type C virus immunoglobin G (IgG). However, the recombinant pET-30VP0 inclusion bodies failed to refold after dialysis process.Many reports confirmed that pocine interferon-α(poINF-α) could elicit immune response and inhibit the replication of FMD virus. In this study, poIFN-αwas expressed in E.coli in order to enhance the immune effect of the candidate vaccine as an immunostimulatory agent. High level of specific antibodies against FMDV type C were elicited in the Balb/C mice inoculated with the mixture recombinant proteins of VP1 and VP3. Experiment results showed that the recombinant poIFN-a failed to enhance the immune effect as expected in the incoluated mice. Whether it could enhance the immune effects in pigs or not needs to be proved.In short, the refolded E. coli-expressed VP1 and VP3 structural proteins of FMDV type C could elicit satisfadtory immune effect, which laid foundation for further preparation of subunite vaccine of FMD type C.