表达大鼠Cdh1基因的重组慢病毒载体的构建及鉴定  

Construction and identification of recombinant lentivirus vector of rat Cdh1 gene

作　　者：邱瑾[1] 姚文龙[1] 钱巍[1] 张传汉[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉科,武汉430030

出　　处：《国际麻醉学与复苏杂志》2012年第3期182-185,共4页International Journal of Anesthesiology and Resuscitation

基　　金：国家自然科学基金资助项目（30872452）

摘　　要：目的构建表达大鼠Cdh1基因的重组慢病毒载体。方法根据大鼠Cdh1基因的核苷酸序列，合成目的基因片段并亚克隆到慢病毒表达载体pGC-FU的AgeI酶切位点间，命名为pGC-FU-Cdh1。对pGC-FU-Cdh1进行PCR扩增及测序鉴定。将293T细胞按完全随机法分为实验组（每组3只）及对照组（每组3只），分别将pGC-FU-Cdh1及空质粒pGC-FU以脂质体法转染至293T细胞，倒置荧光显微镜观察后，Western blot法检测Cdh1-GFP的表达情况，慢病毒载体LV-Cdh1的包装浓缩及滴度测定。结果重组慢病毒表达载体pGC-FU-Cdh1经PCR扩增鉴定及测序鉴定均显示有特异性基因片断，证明大鼠Cdh1基因正向插入慢病毒表达载体pGC-FU中；转染293T细胞后，Westernblot法检测到实验组有外源性融合蛋白Cdh1-GFP的表达（每组3只），对照组无Cdh1-GFP的表达（n=0）（P=0．014）；慢病毒载体LV-Cdh1包装浓缩后，Reahime PCR法测定滴度为2E＋8TU／ml。结论成功构建表达大鼠Cdh1基因的重组质粒pGC-FU-Cdh1，并包装为慢病毒，为进一步研究Cdh1功能及基因治疗奠定了基础。Objective To construct recombinant lentivirus vector of rat Cdhl gene. Methods The artificially synthesized full length DNA of rat Cdhl gene was inserted in the pGC-FU vector, which was isolated by restriction enzyme digest with Age I. The positive recombinant was identified by PCR analysis and sequence analysis. Six culture dishes of 293T cell were randomly allocated into intervention group and control group. Expression of GFP protein was detected by fluorescence microscope and Westen blot in pGC-FU-Cdhl transfected 293T cells. Lentivirus pacaging, concentration and titering were done. Results The PCR analysis and sequence analysis demonstrated that the size and position of Cdhl gene insertion were consistent with the design. Westen blot analysis showed specific expression of external fusion protein of Cdhl-GFP in pGC-FU-Cdhl transfected 293T cells （n=3）, and have no expression in control group （n=0）（P=-0.014）. Lentivirus titering by real-time quantitative PCR was 2E＋8 TU/ml. Conclusions The effective recombinant lentivirus vector of rat Cdhl gene was successfully constructed and may serve as the basis for the further study of Cdhl gene function.

关 键 词：细胞周期蛋白 APC-Cdh1 慢病毒 转基因技术 

分 类 号：R346[医药卫生—基础医学]