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

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.