PRRS病毒主要受体蛋白CD151和CD163真核表达载体的构建  

Construction of Eukaryotic Expression Vector for the CD151 and CD163 Receptor Proteins of Porcine Reproductive and Respiratory Syndrome Virus

作　　者：阮征 李杰 刘开武 王莲芳 华娟 胡小明[3] 刘杰[1,3] 黄海军 

机构地区：[1]武汉市畜牧兽医科学研究所,武汉430208 [2]武汉市重大动物疫病防控中心,武汉430023 [3]华中农业大学猪遗传育种农业部重点实验室&农业动物遗传育种与繁殖教育部重点实验室,武汉430070 [4]武汉市畜牧兽医局,武汉430023

出　　处：《生物技术通报》2014年第12期190-194,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31201938);武汉市科技局项目(2013020705070350-9;2013020705070350-14);武汉市农科院创新项目(CX201309);武汉市农科院青年英才项目(YC201101)

摘　　要：为了进一步研究PRRSV与细胞受体之间的相互作用机理,通过基因工程方法从PAM细胞中克隆获得了CD151和CD163全长编码片段,利用Sal I和Kpn I内切酶对回收得到的CD151(762 bp)和CD163(3 333 bp)DNA片段进行酶切,构建了p IRES2-EGFP-CD151和p IRES2-EGFP-CD163真核表达载体。经PCR和酶切鉴定后,分别提取重组质粒进行细胞转染。荧光显微镜下可见单独转染CD151和CD163基因或CD151/CD163共转染的Marc 145细胞表达强烈的绿色荧光,Western blot分析结果进一步证实,CD151和CD163蛋白在转染后的细胞中获得超表达。猪CD151和CD163真核表达载体的成功构建为进一步探索CD151和CD163在PRRSV感染细胞过程中的协同作用提供了物质基础,特别是对深入研究PRRS病毒的入侵及宿主识别具有重要意义。In order to study the interaction mechanism between the PRRSV and cell receptors, the CD151 and CD163 code fragments were obtained from PAM cells by PCR. These PCR products were digested with Sal I and Kpn I, then inserted into pIRES2-EGFP vectors separately to construct eukaryotic expression plasmid pIRES2 EGFP - CD15I or pIRES2 EGFP - CD163. These vectors were identified by the methods of restriction enzyme digestion, PCR and sequencing. Marc 145 cells transfected with CD15I or CD163 or CD151/CD163 expressed strong green fluorescence, which was further confirmed by western blot. The successful establishment of eukaryotic expression vectors of CD151 and CD163 provides the material foundation for the further exploring the synergistic effect of CD151 and CD163 in the process of PRRSV infection, especially to the further study of PRRS virus invasion and the host identification.

关 键 词：猪繁殖与呼吸综合征病毒 CD151分子 CD163分子 真核表达载体 细胞转染 

分 类 号：S858.28[农业科学—临床兽医学]