人周期素依赖性激酶10真核表达质粒的构建及鉴定  

Construction and identification of eukaryotic expression vector for human cyclin-dependent kinase 10

作　　者：游颜杰[1] 苑艺[1] 李文梅[1] 刘佳佳[1] 张晓辉[2] 

机构地区：[1]漯河医学高等专科学校药学系,河南漯河462000 [2]河南科技大学第一附属医院放疗科,河南洛阳471000

出　　处：《中国生物制品学杂志》2015年第1期43-45,共3页Chinese Journal of Biologicals

基　　金：河南省科技攻关计划项目(142102310464);漯河医学高等专基金项目:科学校自然科学项目(2013;Y-J You)

摘　　要：目的构建人周期素依赖性激酶10(cyclin-dependent kinase 10,CDK10)真核表达质粒,并进行鉴定。方法采用RT-PCR法从人鼻咽癌细胞株HNE1中扩增CDK10基因全长编码区序列,克隆入真核表达载体pc DNA3.1(+)中,构建重组表达质粒pc DNA-CDK10,转染人鼻咽癌细胞株CNE-2,采用Western blot法检测转染细胞中CDK10蛋白的表达水平。结果重组真核表达质粒pc DNA-CDK10经双酶切及测序证实构建正确;经800μg/ml的G418筛选2周后,获得过表达CDK10的稳定转染细胞系CDK10c1和CDK10c2;与空载体转染对照组相比,CDK10c1和CDK10c2细胞中CDK10蛋白的表达量均显著升高(P均<0.05)。结论成功构建了人CDK10真核表达质粒。Objective To construct and identify a eukaryotic expression vector for human cyclin-dependent kinase 10 （CDK10）. Methods The full-length encoding region sequence of CDK10 gene was amplified by RT-PCR from human nasopharyngeal carcinoma NHE1 cells and cloned into eukaryotic expression vector pcDNA3. 1（＋）. The constructed recom- binant plasmid pcDNA-CDK10 was transfected to CNE-2 cells, and the expression level of CDK10 was determined by WesteIn blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pcDNA-CDK10 was constructed correctly. Stable transfected CDK10cl and CDK10c2 cells for over-expression of CDK10 were obtained 2 weeks after screening with 800 μg/ml G418, in which the expression levels of CDK10 increased significantly as compared with those in the cells transfected with empty vector （each P 〈 0. 05 ）. Conclusion The eukaryotic expression vector for human CDK10 was successfully constructed.

关 键 词：人周期素依赖性激酶10 真核细胞 基因表达 鼻咽癌 

分 类 号：Q556[生物学—生物化学] Q782