人真核表达的质粒载体pLenti6/V5-DEST-Cx43的构建及鉴定  

Construction and identification of eukaryotic expressed plasmid vector pLenti6/V5- DEST-Cx43 in human

作　　者：范慧敏[1] 李阳[1] 刘中民[1] 

机构地区：[1]同济大学附属东方医院东方中德心脏中心心外科,上海200120

出　　处：《中华胸心血管外科杂志》2006年第2期130-132,共3页Chinese Journal of Thoracic and Cardiovascular Surgery

基　　金：上海市科技发展基金资助(02ZB14078)

摘　　要：目的构建人真核表达的质粒载体pLenti6/V5-DEST-Cx43,为研究连接蛋白Cx43在心肌细胞间通讯、心脏胚胎发育及心肌细胞分化中的作用机制提供物质基础。方法从质粒p18-T上用限制性内切酶BamH1、Xho1切下Cx43cDNA片断,进行琼脂糖电泳,割胶回收纯化;限制性内切酶BamH1、Xho1酶切质粒pENTR11,将线形化的载体与回收的Cx43cDNA片断用T4DNA连接酶连接,测序后通过LR反应克隆到慢病毒载体pLenti6/V5-DEST中。结果凝胶电泳和测序结果均证明已将人类Cx43cDNA克隆到慢病毒载体pLenti6/V5-DEST中。结论成功构建了人真核表达的质粒载体pLenti6/V5-DEST-Cx43。Objective To construct eukaryotic expressed plnsmid vector pLenti6/V5- DEST-Cx43 in human for further study on bioinfonnative communicatien between myocardial cell, embryonic development of heart and differentiation of myocardial cell. Methotis Cx43 cDNA fragments were obtained from plasmid p18-T by restriction enzyme BamH1 ,Xhol, after the agarese gel electrophoresis was performed, the Cx43 cDNA was retrieved, pENTRll were extracted by enzyme BamH1 ,Xho1, then connected with Cx43 cDNA by T4 DNA ligase. LR reaction was perfenned and Cx43 cDNA were cloned into Lentiviral vector pLenti6/V5-DEST. Results Agarese electrophoresis and sequent examination, identified that Cx43 cDNA was cloned into Lentiviral vector pLenti6/V5-DEST. Conclusion The present experiment demonstrate a succesful cloning of human Cx43 cDNA into the Lentiviral vector pLenti6/V5- DEST.

关 键 词：连接蛋白43 慢病毒属 真核细胞 基因表达 

分 类 号：R346[医药卫生—基础医学]