关键词: 信号肽, E2蛋白, 猪瘟病毒(CSFV), 分泌表达

Abstract: This study aimed to achieve the effective secretory expression of E2 of classical swine fever virus (CSFV) using baculovirus by introducing the signal peptide. Firstly, the pFastBac1 vector was modified by adding the endogenous E2 signal peptide, the melittin signal peptide or the gp67 signal peptide, respectively. Then the E2 gene fragment was cloned into the modified vectors. The resulting plasmids were then transformed into the DH10Bac competent cells to get recombinant bacmids through blue-white screening. The bacmids were subsequently transfected into the sf21 cells to generate recombinant baculoviruses. Secondly, the sf21 cells were infected for large-scale protein production. The E2 protein was purified by nickel-affinity chromatography and the purity was identified using SDS-PAGE. Finally, the glycosylation pattern and the immunogenicity of the purified E2 were analyzed. Results were as follows:Consequently, the recombinant plasmids were generated. The recombinant bacmids were acquired through blue-white screening. The Western blot analysis showed that the secretory expression of E2 was achieved by introducing signal peptides. Among the three signal peptides, the gp67 mediated the highest secretion efficiency. It was shown that the E2 protein was pure and the yield was about 25 mg·L^-1. Indirect ELISA results demonstrated that the purified E2 protein could be specifically recognized by the CSFV positive serum, indicating that the purified E2 protein was immunogenic. The purified E2 protein was used to immunize BALB/c mice after emulsification with ISA201 adjuvant. The blocking ELISA result showed that the antibody could be detected after 14 days of immunization, indicating that the E2 protein had good immunogenicity. Taken together, the study is useful for developing a subunit vaccine against CSFV using the E2 expressed in the sf21 cells.

Key words: signal peptide, E2 protein, classical swine fever virus (CSFV), secretory expression