Construction Of Virus-like Particles Of Structural Protein VP1of Foot-and-mouth Disease Virus And Its Immunogenicity

FMD (foot-and-mouth disease, FMD) is an acute, severe, highly contagious viral disease, whichcaused by Foot-and-mouth disease virus (FMDV). The virus is a member of the Aphthovirus in the familyPicornaviridae, which affects animals with cloven hoofed animals, such as cattle, sheep, goats, pigs, anddeer. In addition to the direct cause of the pup mortality, FMD reduces body weight and milk production,and leads to international trade restrictions of animals and animal products around the world. Consideringserious economic losses, FMD is the greatest threat of infectious diseases to cloven-hoofed animals. So far,the whole virus inactivated vaccine is the most effective tool to prevent and control FMD. However, thereare many deficiencies in the vaccine which is widely used in currently. At now, it is extremely urgent todevelop some new vaccines to address these deficiencies.There are important B-cell and T-cell epitopes involved in FMDV structural protein VP1, whichconstitute a major antigenic sites. Therefore, we constructed a double-layer structure virus-likeparticles(VLPs) which is assembled by a fusion protein P14-VP1, and its immune effect to mice wasstudied too.The sequence of Bovine viral diarrhea virus(BVDV)JZ05-1strain, the169~270amino acids ofpolyprotein, its core protein P14, and the1~211amino acids of polyprotein P1of FMDV Asia1/JS/CHA/05, its structural protein VP1, were selected out, respectively. Between the two peptides, aflexible motif sequence was inserted into as a linker. The P14-linker-VP1coding sequence was amplifiedby fusion PCR, fused and inserted into pBLR18which contains the signal peptide, to construct arecombinant plasmid pBLR18-P14-VP1. Then, the signal peptide-P14-VP1gene was cloned into Bacillussubtilis expression vector p7257-P43to build up the shuttle plasmid p7257-P43-P14-VP1, and its positiveclones were screened out, and confirmed by sequence analysis. Subsequently, the shuttle plasmid wastransformed into Bacillus subtilis WB800cells, and SDS-PAGE analysis showed that expression productconsists in the WB800cells in LB broth with40μg/mL chloromycetin (Cm), and the fusion proteinP14+VP1could specifically react with the bovine positive sera against FMDV type Asia1in Westernblotting test. By electron microscopy observation, the fusion protein can produce VLPs, which diameter is50nanometers aproximately.The P14-VP1fusion protein expressed by WB800was assembled into VLPs in vitro.The health micewere immunized intramuscularly two times with the fusion protein, and set inactivated FMDV and PBS ascontrol group. The serum was separated0,2,4,6,8,10,12,14and16weeks after immunization,respectively. Subsequently, indirect ELISA and virus neutralization test was performed for detection ofhumoral immunity. Compared with the control groups, mice inoculated with the fusion protein couldproduce the ELISA IgG, but neutralizing antibody could not be detected out.