稳定高效表达猪CD163的Marc-145细胞系的构建与鉴定

doi:10.16431/j.cnki.1671-7236.2015.08.022

›› 2015, Vol. 42 ›› Issue (8): 2074-2080.doi: 10.16431/j.cnki.1671-7236.2015.08.022

稳定高效表达猪CD163的Marc-145细胞系的构建与鉴定

吴香菊^1,2, 李芳韬¹, 陈蕾^2,3, 丛晓燕^2,3, 孙文博^2,3, 于江^2,3, 陈智^2,3, 郭立辉^2,3, 吴家强^2,3, 杜以军^2,3, 赵孝民¹, 王金宝²

1. 山东农业大学动物科技学院, 山东省动物生物工程与疾病防治重点实验室, 泰安 271000;
2. 山东省畜禽疫病防治与繁育重点实验室, 济南 250100;
3. 山东省农业科学院畜牧兽医研究所, 济南 250100

收稿日期:2015-01-21 出版日期:2015-08-20 发布日期:2015-08-27
通讯作者: 杜以军, 赵孝民, 王金宝 E-mail:duyijun0916@163.com;xmzhao66@163.com;wangjb@saas.ac.cn
作者简介:吴香菊(1990-),女,山东潍坊人,硕士生,研究方向:流行病及病原分子生物学,E-mail:wuxiangju900426@163.com
基金资助:
山东省自然科学杰出青年基金(JQ201411);国家自然科学基金(31170146、31370189);公益性行业(农业)科研专项(201303046);中国博士后科学基金特别资助项目(2014T70645);山东省优秀中青年科学家科研奖励基金(BS2012NY012);山东省现代农业产业技术体系(SDAIT-06-022-06);山东省农业科学院科技创新重点项目(2014CXZ08-2、2014CGPY04);山东省农业科学院青年英才培养计划经费资助

Establishment and Identification of One Marc-145 Cell Line Stably and Highly Expressing Porcine CD163

WU Xiang-ju^1,2, LI Fang-tao¹, CHEN Lei^2,3, CONG Xiao-yan^2,3, SUN Wen-bo^2,3, YU Jiang^2,3, CHEN Zhi^2,3, GUO Li-hui^2,3, WU Jia-qiang^2,3, DU Yi-jun^2,3, ZHAO Xiao-min¹, WANG Jin-bao²

1. Key Laboratory of Animal Biotechnology and Disease Control and Prevention of Shandong Province, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271000, China;
2. Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan 250100, China;
3. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China

Received:2015-01-21 Online:2015-08-20 Published:2015-08-27

摘要/Abstract

摘要： 试验旨在构建一株高效表达猪CD163(pCD163)的Marc-145细胞系,为猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的临床分离和疫苗生产奠定基础。根据GenBank中序列设计引物从猪肺泡巨噬细胞(PAM)中扩增pCD163基因,将其插入真核表达载体pCI-neo构建真核表达质粒pCI-pCD163,将该重组质粒转染Marc-145细胞,通过G418筛选、单克隆化并扩大培养筛选获得表达pCD163的Marc-145细胞系,IFA、Western blotting鉴定其表达情况。IFA结果显示,构建的pCD163-Marc细胞系中荧光明显亮于普通Marc-145细胞;Western blotting结果显示,pCD163-Marc细胞系中CD163蛋白表达量约为对照Marc-145细胞中CD163蛋白表达量的8.7倍。且该细胞系可稳定传至20代,各代次之间表达量无差异。证明高效表达猪CD163的Marc-145细胞系构建成功。

关键词: Marc-145; CD163; 稳定细胞系

Abstract: This study was attempted to generate one Marc-145 cell line stably and highly expressing porcine CD163 (pCD163) and set the foundation for PRRSV isolation and vaccine production.CD163 was shown to be a cellular receptor capable of mediating infection of PRRSV non-permissive cell lines.The pCD163 gene was amplified by RT-PCR from porcine alveolar macrophages and cloned into the eukaryotic expression vector pCI-neo, then the positive plasmid pCI-pCD163 was transfected into Marc-145 cells.After selecting with G418 and subcloning for 3 times, Marc-145 cell line expressing pCD163 was established.IFA results indicated that the fluorescence of pCD163-Marc cells was significantly brighter than Marc-145 cells;Western blotting results indicated that the pCD163-Marc cells could express higher levels of CD163 and the expression level was 8.7 times higher than Marc-145 cells.The pCD163-Marc cell line could be stably passaged for 20 passages and the expression level of CD163 was similar with different passages, which would be a valuable tool for facilitating virus propagation and vaccine production.

Key words: Marc-145; CD163; stable cell line

中图分类号:

Q813

吴香菊, 李芳韬, 陈蕾, 丛晓燕, 孙文博, 于江, 陈智, 郭立辉, 吴家强, 杜以军, 赵孝民, 王金宝. 稳定高效表达猪CD163的Marc-145细胞系的构建与鉴定[J]. , 2015, 42(8): 2074-2080.

WU Xiang-ju, LI Fang-tao, CHEN Lei, CONG Xiao-yan, SUN Wen-bo, YU Jiang, CHEN Zhi, GUO Li-hui, WU Jia-qiang, DU Yi-jun, ZHAO Xiao-min, WANG Jin-bao. Establishment and Identification of One Marc-145 Cell Line Stably and Highly Expressing Porcine CD163[J]. , 2015, 42(8): 2074-2080.

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稳定高效表达猪CD163的Marc-145细胞系的构建与鉴定

Establishment and Identification of One Marc-145 Cell Line Stably and Highly Expressing Porcine CD163

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