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.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
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)~~
关键词
NOV基因
RT-PCR
基因克隆
nephroblastoma overepression gene
RT-PCR
gene cloning