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

作　　者：吴香菊[1,2] 李芳韬 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] 

机构地区：[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

出　　处：《中国畜牧兽医》2015年第8期2074-2080,共7页China Animal Husbandry & Veterinary Medicine

基　　金：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

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pCIpCD163,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。This study was attempted to generate one Marc-145 cell line stably and highly express- ing 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-permis- sive 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 indica- ted 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-Mare 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.

关 键 词：MARC-145 CD163 稳定细胞系 

分 类 号：Q813[生物学—生物工程]