Expression and optimization of the structural protein VP1 of foot-and-mouth disease virus in Corynebacterium glutamicum

Abstract:

[Background] Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the major diseases that infect cloven-hoofed animals such as cattle, sheep, and pigs. The structural protein VP1 of FMDV contains multiple sites that can cause host immune response, serving as a target for the research and development of subunit vaccines. As a safe production strain, Corynebacterium glutamicum is an elite cell factory for the production of pharmaceutical proteins. [Objective] To realize the heterologous expression of VP1 in C. glutamicum. [Methods] According to the gene sequence and function of VP1 and the codon preference of C. glutamicum, we designed and synthesized the VP1 gene and then ligated it with pXMJ19 to create the recombinant plasmid pXMJ19-VP1. C. glutamicum CGMCC 1.15647 was used to express VP1 protein. The protein expression elements [e.g., the promoter, 5′ untranslated region (5′UTR), and N-terminus] of VP1 protein and the culture conditions were optimized. SDS-PAGE and Western blotting were employed to determine the expression of VP1. Indirect enzyme-linked immunosorbent assay (ELISA) was employed to measure the immunocompetence of VP1 produced in this study. [Results] SDS-PAGE and Western blotting showed that VP1 was successfully expressed in C. glutamicum CGMCC 1.15647. Replacing P_tac promoter with the synthetic promoter P_H36 increased the protein yield. On this basis, the protein yield can be further increased by insertion of the 5'UTR sequence and mutation of the N-terminal amino acid of VP1. CspB signal peptide can be used to achieve secretory expression. Fermentation experiments showed that the optimum conditions of shake flask fermentation were 30 ℃ and 24 h. The results of ELISA suggested that VP1 could specifically bind to the positive serum. [Conclusion] The VP1 protein of FMDV was successfully expressed in C. glutamicum CGMCC 1.15647, and optimizing protein expression elements increased the protein yield, which laid a foundation for the development of immunodiagnostic reagents and safe and efficient subunit vaccines for FMD.

Key words:Corynebacterium glutamicum;foot-and-mouth disease virus;structural protein VP1;protein expression element;fermentation optimization