| 稳定高效表达猪CD163的Marc-145细胞系的构建与鉴定 |
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| 引用本文： | 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].中国畜牧兽医,2015,42(8):2074-2080. |
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| 作者姓名： | 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 |
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| 作者单位： | 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 |
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| 基金项目： | 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 |
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| 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。
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| 关 键 词： | Marc-145 CD163 稳定细胞系 |
| 收稿时间： | 2015-01-21 |
Establishment and Identification of One Marc-145 Cell Line Stably and Highly Expressing Porcine CD163 |
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| 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].China Animal Husbandry & Veterinary Medicine,2015,42(8):2074-2080. |
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| Authors: | 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 |
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| Institution: | 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 |
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| 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. |
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| Keywords: | Marc-145 CD163 stable cell line |
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