HSP27慢病毒载体的构建及其稳定表达细胞株的建立  

Construction of HSP27 lentiviral vector and establishment of its stable expression cell lines

作　　者：马瑞仙 牛宇辉 吴贝 李向茸[1,2] 许淑娟 李琼毅[1,3] 柏家林 冯若飞[1,2] MA Rui-xian;NIU Yu-hui;WU Bei;LI Xiang-rong;XU Shu-juan;LI Qiong-yi;BAI Jia-lin;FENG Ruo-fei(Key Laboratory of Biotechnology&Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;Biomedical Research Center,Northwest Minzu University,Gansu Animal Cell Technology Innovation Center,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030,China)

机构地区：[1]西北民族大学生物医学研究中心,生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]西北民族大学生物医学研究中心,甘肃省动物细胞技术创新中心,甘肃兰州730030 [3]西北民族大学,生命科学与工程学院,甘肃兰州730030

出　　处：《中国兽医科学》2021年第1期105-112,共8页Chinese Veterinary Science

基　　金：西北民族大学中央高校基本科研业务费资金项目(31920190003,31920200003);国家民委中青年英才培养计划项目[批准号(2018)98];教育部“创新团队发展计划”项目(IRT_17R88)。

摘　　要：热休克蛋白27(HSP27)是一种结构上高度保守的小分子热休克应激蛋白,它可通过与细胞自噬、细胞凋亡和天然免疫信号通路关键因子相互结合参与多种病毒的感染过程。为了便于深入研究HSP27在机体抗感染免疫中的作用,用PCR方法从A549细胞中扩增出HSP27片段,然后经双酶切、连接、转化等步骤获得HSP27的慢病毒表达载体pTRIP-HSP27,将此载体和另一对照载体pTRIP-EGFP分别转染至HEK-293T细胞中,48 h后收集慢病毒将其转导至A549细胞,经嘌呤霉素筛选获得过表达HSP27的细胞株A549-HSP27和过表达EGFP的对照细胞株A549-EGFP。最后,经RT-qPCR、Western-blottin、gIFA和细胞活力检测等方法进行鉴定,获得了能稳定表达HSP27的细胞株。Heat shock protein 27(HSP27)is a small heat shock stress protein with highly conserved structure.It can participate in the infection process of various viruses by interacting with key factors of autophagy,apoptosis and innate immune signaling pathways.In order to further study the role of HSP27 in anti-virus immunity,HSP27 was amplified from A549 cells by PCR,and then the recombinant lentiviral expression vector p TRIP-HSP27 was obtained by double enzyme digestion,ligation and transformation.Then,p TRIP-HSP27 and the control vector p TRIP-EGFP were transfected into HEK-293 T cells respectively.After 48 hours,lentivirus was collected and transferred to A549 cells.Finally,these stable cell lines A549-HSP27 and A549-EGFP were identified by RT-q PCR,Western-blotting,IFA and cell viability assay.

关 键 词：热休克蛋白27 慢病毒 稳定细胞株 

分 类 号：S852.65[农业科学—基础兽医学]