CHO细胞表达牛病毒性腹泻病毒E2蛋白及免疫原性分析

doi:10.11843/j.issn.0366-6964.2022.12.017

Abstract

Abstract: This experiment was conducted to produce E2 protein of bovine viral diarrhea virus (BVDV) by using suspension cultured CHO cell expression system, and to identify the immunogenicity of purified E2 protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E2 was constructed based on the gene sequence of BVDV-1 NADL strain. The recombinant plasmid pcDNA3.1-BVDV-E2 was transfected into CHO cells in suspension culture, and the E2 protein was secreted and expressed in cells supernatant. The expression and purification of the E2 protein was determined by SDS-PAGE electrophoresis, and the reactivity was determined with anti-His antibody and BVDV positive serum by Western blot. Analysis of immunogenicity was determined after immunizing New Zealand white rabbits with E2 protein, and the antibodies of the E2 protein were tested by indirect ELISA and indirect immunofluorescence (IFA). The concentration of purified E2 protein was 1.228 mg·mL^-1 by BCA protein quantification kit. Western blot results showed that specific bands of the E2 protein could be detected with His antibody and BVDV positive serum. Serum antibody could be detected by indirect ELISA in the 7th day after prime immunization, and serum antibody was maintained at a high level until the 28th day after immunization. The antibody titer was up to 1:1 024 000. The result of IFA indicated that the expression of the E2 protein could be detected by the immunized rabbit serum in BVDV-infected MDBK cells. These results demonstrated that the purified E2 protein with good immunogenicity and specificity. In this study, BVDV E2 protein was produced using CHO suspension culture system successfully, which has a superior immunogenicity. This result will lay the foundation for the development of diagnosis method and novel subunit vaccine of BVDV.

Key words: bovine viral diarrhea virus, E2 protein, eukaryotic expression, CHO suspension culture, immunogenicity

CLC Number:

S852.659.6

LI Yajun, RU Yi, HAO Rongzeng, JIANG Chenghui, WANG Wei, ZHANG Yue, ZHANG Guicai, LIU Huanan, LU Bingzhou, YANG Yang, TAO Shiyu, YANG Rui, SONG Xiangdong, CHEN Jiao, YU Sijiu, ZHENG Haixue. Expression of Bovine Viral Diarrhea Virus E2 Protein in CHO Cells and Immunogenicity Analysis[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4315-4324.

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Expression of Bovine Viral Diarrhea Virus E2 Protein in CHO Cells and Immunogenicity Analysis

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