NOV基因真核表达载体的构建及在COS-7细胞中的表达  

Construction of eukaryotic expression vectors of nephroblastoma overexpression gene and expression in COS-7 cells

作　　者：李成仁[1] 蔡文琴[1] 苏炳银[1] 张成岗[1] 

机构地区：[1]第三军医大学基础医学部组织学与胚胎学教研室,重庆市神经科学研究所,重庆400038

出　　处：《第三军医大学学报》2005年第12期1187-1189,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目 ( 3990 0 0 78) ;全军医学科研"十五"计划青年基金资助项目 ( 0 1Q0 97) ;重庆市科委应用基础研究资助项目( 2 0 0 1)~~

摘　　要：目的　获得NOV基因真核表达载体并检测其在细胞中的表达。方法　利用逆转录 多聚酶链反应(RT PCR)方法,从新鲜大鼠大脑组织的总cDNA中扩增出1165bp的NOV基因的cDNA编码区序列,然后用HindⅢ和BamHⅠ双酶切后定向克隆入真核表达载体pcDNA3 1 Myc His(+ ) lacZ质粒中,用脂质体法将载体转染入COS 7细胞中,用Westernblot和免疫细胞化学方法检测其表达情况。结果　NOV基因cDNA已经正确克隆到真核表达载体pcDNA3 .1 Myc His(+ ) lacZ质粒中;体外转染入COS 7细胞中后,可见转染细胞有NOV蛋白的表达。结论　本实验所构建的重组质粒为NOV基因的作用研究提供了有利的分子工具。Objective To obtain eukaryotic expression vectors containing coding region of nephroblastoma overexpression gene (NOV) and detect its expression in COS-7 cells. Methods A 1 165-bp cDNA fragment was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1/Myc-His(+)/lacZ. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes HindⅢ and BamHⅠ. The recombinant plasmid was transfected into COS-7 cells with liposome. The expression of NOV gene was detected by Western blotting and immunocytochemistry. Results Eukaryotic expression vectors containing 1 165 -bp coding region of NOV gene was constructed. COS-7 cells transfected with the recombinant plasmid expressed high level of NOV protein in cytoplasm. Conclusion That eukaryotic expression vectors containing coding region of NOV gene was constructed can provide a strong molecular tool for the studies of effect of NOV gene.

关 键 词：NOV基因 RT-PCR 基因克隆 

分 类 号：Q782[生物学—分子生物学] R394.33[医药卫生—医学遗传学]