稳定表达猪圆环病毒2型Cap蛋白的CHO-K1细胞系的建立及免疫原性分析

doi:10.11843/j.issn.0366-6964.2019.05.016

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (5): 1056-1063.doi: 10.11843/j.issn.0366-6964.2019.05.016

稳定表达猪圆环病毒2型Cap蛋白的CHO-K1细胞系的建立及免疫原性分析

武乐祎^1,2,3, 吴素芳³, 车影³, 张强³, 李鹏¹, 卞广林^3*, 王选年^1*

1. 新乡学院生命科学与技术学院生物技术研究中心, 新乡 453003;
2. 郑州大学, 郑州 450000;
3. 浙江海隆生物技术有限公司, 绍兴 312000

收稿日期:2018-08-28 出版日期:2019-05-23 发布日期:2019-05-23
通讯作者: 王选年(1969-),主要从事动物病理学和分子免疫学研究,E-mail:wangxuannian@163.com;卞广林(1976-),主要从事免疫学研究,E-mail:g.bian@novo-biotech.com
作者简介:武乐祎(1993-),女,河南洛阳人,硕士,主要从事分子免疫学研究,E-mail:616961225@qq.com
基金资助:
国家重点研发计划（2016YFD0500702）；河南省科技攻关项目（122102110142）；河南省高等学校重点科研项目（19A230009）

The Establishment of CHO-K1 Cell Line Stably Expressing High Level PCV2-Cap and Immunogenicity Analysis

WU Leyi^1,2,3, WU Sufang³, CHE Ying³, ZHANG Qiang³, LI Peng¹, BIAN Guanglin^3*, WANG Xuannian^1*

1. Biotechnology Research Center, College of Life Science and Technology, Xinxiang University, Xinxiang 453003, China;
2. Zhengzhou University, Zhengzhou 450000, China;
3. Zhejiang Novo-biotech Co., Ltd., Shaoxing 312000, China

Received:2018-08-28 Online:2019-05-23 Published:2019-05-23

摘要/Abstract

摘要：

建立高表达PCV2-Cap蛋白的CHO-K1悬浮稳转细胞系，鉴定蛋白的免疫原性，为开发新型且有效防治猪圆环病毒的亚单位疫苗奠定基础。构建重组质粒pEE12.4-PCV2-Cap，转染CHO-K1细胞，通过加压筛选，有限稀释，细胞悬浮驯化及Western blot检测得到高表达Cap蛋白的悬浮稳转单克隆细胞株，并对该细胞株进行发酵，纯化得到的目的蛋白，通过小鼠免疫验证其免疫原性。结果表明PCV2-Cap蛋白能够在CHO-K1细胞中正确表达；发酵过程中活细胞密度高达6×10⁶个·mL^-1，细胞活力在80%以上，PCV2-Cap蛋白表达量约为370 mg·L^-1；利用间接ELISA检测小鼠免疫后血清中的抗体水平，证明了利用CHO-K1细胞生产的Cap蛋白具备良好的免疫原性。本研究成功构建了表达PCV2-Cap蛋白的CHO-K1悬浮稳转细胞系，并进一步对目的蛋白进行免疫原性分析，为猪圆环病毒亚单位疫苗的开发打下扎实的基础。

Abstract:

In this study, we established a Chinese Hamster Ovary (CHO-K1) cell line stably expressing PCV2-Cap, and identified the immunogenicity of the protein and prepared for a kind of novel subunit vaccine against porcine circovirus. Firstly, we constructed the recombinant plasmid pEE12.4-PCV2-Cap. Then we transfected adherent CHO-K1 cells with the plasmid. To obtain the monoclonal cell line, the transfected cells were selected with the methods of pressurized screening and limited dilution. And then the monoclonal cell was optimized for growth in suspension and cultured in fed-batch mode for the quality evaluation. The cell strain was fermented, and the purified target protein was verified by immunoassay in mice. The results demonstrated that PCV2-Cap protein was correctly expressed by CHO-K1 cells. The maximum density of living cells reached 6×10⁶ per milliliter, the cell viability was more than 80% in the process of the culture, the PCV2-Cap production can get to 370 mg·L^-1. The results of ELISA showed that Cap protein produced by CHO-K1 has good immunogenicity. A CHO-K1 cell line stably expressing PCV2-Cap protein has been established, and the immunogenicity analysis of the target protein was further carried out. It lays a foundation for the development of new porcine circovirus subunit vaccine.

中图分类号:

S852.659.2

武乐祎, 吴素芳, 车影, 张强, 李鹏, 卞广林, 王选年. 稳定表达猪圆环病毒2型Cap蛋白的CHO-K1细胞系的建立及免疫原性分析[J]. 畜牧兽医学报, 2019, 50(5): 1056-1063.

WU Leyi, WU Sufang, CHE Ying, ZHANG Qiang, LI Peng, BIAN Guanglin, WANG Xuannian. The Establishment of CHO-K1 Cell Line Stably Expressing High Level PCV2-Cap and Immunogenicity Analysis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1056-1063.

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稳定表达猪圆环病毒2型Cap蛋白的CHO-K1细胞系的建立及免疫原性分析

The Establishment of CHO-K1 Cell Line Stably Expressing High Level PCV2-Cap and Immunogenicity Analysis

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