Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis

Abstract:

The aim of this study was to produce E^rns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified E^rns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E^rns was constructed based on the gene sequence of BVDV-1 NADL strain. The E^rns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-E^rns into CHO cells. The expression and purification of the E^rns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the E^rns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified E^rns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the E^rns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the E^rns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log₁₀ was induced in rabbits. In this study, purified BVDV E^rns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.