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

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (12): 4315-4324.doi: 10.11843/j.issn.0366-6964.2022.12.017

CHO细胞表达牛病毒性腹泻病毒E2蛋白及免疫原性分析

李亚军^1,2, 茹毅², 郝荣增², 蒋成辉², 王伟², 张越², 张贵才², 刘华南², 卢炳州², 杨洋², 陶世宇², 杨锐², 宋向东², 陈娇², 余四九^1*, 郑海学^2*

1. 甘肃农业大学动物医学院, 兰州 730070;
2. 中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室/国家口蹄疫参考实验室, 兰州 730046

收稿日期:2022-03-10 出版日期:2022-12-23 发布日期:2022-12-25
通讯作者: 余四九，主要从事兽医产科学研究，E-mail:sjyu@163.com；郑海学，主要从事动物传染病学与流行病学研究，E-mail:zhenghaixue@caas.cn
作者简介:李亚军(1989-)，男，甘肃秦安人，博士生，主要从事动物新型疫苗研发，E-mail:liyajun073@163.com
基金资助:
甘肃省科学技术厅-科技重大专项计划(21ZD3 NA001)；中国农业科学院兰州兽医研究所基本科研业务费(1610312021013)

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

LI Yajun^1,2, RU Yi², HAO Rongzeng², JIANG Chenghui², WANG Wei², ZHANG Yue², ZHANG Guicai², LIU Huanan², LU Bingzhou², YANG Yang², TAO Shiyu², YANG Rui², SONG Xiangdong², CHEN Jiao², YU Sijiu^1*, ZHENG Haixue^2*

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2. State Key Laboratory of Veterinary Etiological Biology/National Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China

Received:2022-03-10 Online:2022-12-23 Published:2022-12-25

摘要/Abstract

摘要： 旨在利用悬浮培养CHO细胞表达系统制备牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV) E2蛋白，并鉴定纯化E2蛋白的免疫原性。本研究以BVDV-1 NADL株基因序列为基础，构建BVDV E2蛋白的重组真核表达质粒pcDNA3.1-BVDV-E2，转染经悬浮培养的CHO细胞进行分泌表达，收集细胞培养上清，进行亲和层析法纯化，SDS-PAGE电泳鉴定蛋白表达和纯化，Western blot鉴定与His抗体和BVDV阳性血清反应性，利用纯化蛋白免疫新西兰大白兔，用细胞间接免疫荧光(IFA)和ELISA鉴定E2蛋白的反应活性。纯化E2蛋白经BCA蛋白定量试剂盒检测后表达量高达1.228 mg·mL^-1；Western blot结果显示，利用His抗体和BVDV阳性血清均可检测到目的蛋白的特异性条带；新西兰大白兔一免后第7天利用间接ELISA可检测到血清抗体阳性，并持续至免疫后第28天，血清抗体效价水平达到1∶1 024 000；IFA试验结果显示，该血清抗体可检测到BVDV感染MDBK细胞中E2蛋白的表达，进一步证实纯化的E2蛋白具有良好的免疫原性和特异性。本研究成功利用CHO悬浮培养真核表达系统制备了纯化的BVDV E2蛋白，该蛋白具有良好的免疫原性，为BVDV的诊断方法及其新型亚单位疫苗研制奠定了基础。

关键词: 牛病毒性腹泻病毒, E2蛋白, 真核表达, CHO悬浮培养, 免疫原性

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

中图分类号:

S852.659.6

李亚军, 茹毅, 郝荣增, 蒋成辉, 王伟, 张越, 张贵才, 刘华南, 卢炳州, 杨洋, 陶世宇, 杨锐, 宋向东, 陈娇, 余四九, 郑海学. CHO细胞表达牛病毒性腹泻病毒E2蛋白及免疫原性分析[J]. 畜牧兽医学报, 2022, 53(12): 4315-4324.

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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CHO细胞表达牛病毒性腹泻病毒E2蛋白及免疫原性分析

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

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