| CHO细胞表达牛病毒性腹泻病毒E2蛋白及免疫原性分析 |
| |
| 引用本文： | 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. |
| |
| 作者姓名： | 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 |
| |
| 作者单位： | 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 |
| |
| 基金项目： | 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) |
| |
| 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 |
| 收稿时间： | 2022-03-10 |
Expression of Bovine Viral Diarrhea Virus E2 Protein in CHO Cells and Immunogenicity Analysis |
| |
| 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. |
| |
| Authors: | 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 |
| |
| Institution: | 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 |
| |
| 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. |
| |
| Keywords: | bovine viral diarrhea virus E2 protein eukaryotic expression CHO suspension culture immunogenicity |
|
| 点击此处可从《畜牧兽医学报》浏览原始摘要信息 |
| 点击此处可从《畜牧兽医学报》下载免费的PDF全文 |
|
