稳定表达猴源波形蛋白的BHK-21细胞系的建立  

Establishment of a stable BHK-21 cell line expressing vimentin of Marc-145 cell

作　　者：马晓春[1] 高继明[1] 孔宁[1] 高丽丽[1] 王彤彤[1] 肖一红[1] 周恩民[2] 

机构地区：[1]山东农业大学动物医学院,山东泰安271018 [2]西北农林科技大学兽医免疫学研究所/动物医学院,陕西杨凌712100

出　　处：《中国兽医科学》2012年第11期1179-1183,共5页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(U0931003/L01)

摘　　要：为建立稳定表达猴源波形蛋白的BHK-21细胞系,采用RT-PCR方法从非洲绿猴肾细胞(Marc-145)中扩增波形蛋白基因,并将其与EmGFP基因、IRES基因共同克隆到pcDNA3.1/V5-His A载体中,成功构建重组质粒pcDNA3.1-GIV。通过非脂质体转染将其转染至BHK-21细胞中,经G418加压筛选,单克隆挑取,获得稳定表达猴源波形蛋白的细胞系BHK-21-GIV。经Western-blot和RT-PCR检测,证明外源波形蛋白成功得到表达。本研究建立的稳定表达猴源波形蛋白的细胞系BHK-21-GIV,为研究波形蛋白的生物学功能及与猪繁殖与呼吸综合征病毒感染细胞的关系提供了有利的工具。In order to establish a stable BHK-21 cell line expressing vimentin of Marc-145 cell,the vi- mentin gene was amplified by RT-PCR from Marc-145 cells and inserted into pcDNA3.1/V5-His A plasmid together with EmGFP gene and IRES to construct recombinant plasmid pcDNA3. 1-GIV. The pcD- NA3.1-GIV plasmid was transfected into BHK-21 cell to establish stably expressing vimentin with the presence of G418 and then subcloned. Western-blot and RT-PCR results indicated that the BHK-21-GIV cells could express exogenous vimentin,which demonstrated that a cell line stably expressing vimentin was obtained, and designated as BHK-21-GIV. This study provided a useful tool for vimentin studying and provided evidence on the relationship between vimentin and porcine reproductive and respiratory syndrome virus.

关 键 词：波形蛋白 稳定细胞系 BHK-21细胞 猪繁殖与呼吸综合征病毒 

分 类 号：S852.659.6[农业科学—基础兽医学]