哺乳动物细胞CDK2系列表达载体的构建与表达  

Construction and expression of the CDK2 mammalian cell expression vectors

作　　者：吴健谊[1] 蒋太峰[1] 谢剑君[1] 张锴[2] 杜则澎[3] 许丽艳[3] 

机构地区：[1]汕头大学医学院生物化学教研室,广东汕头515041 [2]南开大学化学学院,天津300071 [3]汕头大学医学院肿瘤病理研究室,广东汕头515041

出　　处：《癌变．畸变．突变》2011年第4期245-249,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金青年科学基金项目(31000347);国家自然科学基金-广东联合基金项目(U0932001)

摘　　要：目的:构建一系列细胞周期蛋白依赖激酶2(cyclin-dependent kinase 2,CDK2)在哺乳动物细胞的表达载体,为研究CDK2的功能和修饰提供实验材料。方法:从食管癌细胞中提取总RNA,逆转录PCR扩增CDK2编码区,然后将PCR产物克隆到T载体;扩增后的CDK2片段分别亚克隆入pcDNA3、pcDNA4、pNTAP和pEGFP等4种哺乳动物表达载体;最后,将获得的表达载体PEGFP/CDK2转染小鼠成纤维细胞NIH3T3进行初步的CDK2表达分析。结果:RT-PCR扩增获得约900 bp的目的片段,经T载体克隆和DNA序列分析,显示重组片段是人CDK2基因序列;CDK2片段分别亚克隆入上述4种载体后获得相应表达载体;运用构建的PEGFP/CDK2表达载体,在NIH3T3细胞中表达出CDK2蛋白。结论:成功构建了CDK2的哺乳动物细胞系列表达载体,并在NIH3T3细胞中成功表达目的蛋白。OBJECTIVE：To construct a series of cyclin-dependent kinase 2（CDK2） mammalian cell expression vectors and to assess CDK2 expression in NIH3T3 cells.METHODS：RNA was isolated from esophageal cancer cells and the full coding sequence of CDK2 gene was obtained by RT-PCR.The PCR product was then cloned into T vector and subsequently subcloned into four eukaryotic expression vectors（pcDNA3,pcDNA4,pNTAP and pEGFP）. The expressing plasmids were transfected into NIH3T3 cells and the expression of CDK2 was detected by western blot. RESULTS：The PCR product was about 900 bp and the sequence analysis showed that it was the full coding sequence of CDK2 gene.The product was subcloned into the eukaryotic expression vectors and four CDK2 expression vectors were constructed.Western blot showed that CDK2 could be expressed in the expression vector-transfected cells. CONCLUSION：Four CDK2 eukaryotic expression vectors were successfully constructed and CDK2 was effectively expressed in NIH3T3 cells.

关 键 词：CDK2 基因克隆 真核表达载体 

分 类 号：Q71[生物学—分子生物学] R73-3[医药卫生—肿瘤]