哺乳动物细胞CDK3系列表达载体的构建与表达  

Construction and Expression of the CDK3 Mammalian Cell Expression Vectors

作　　者：吴健谊[1,2] 蒋太峰[1,2] 许丽艳[2] 李恩民[1] 谢剑君[1] 

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]汕头大学医学院病理学教研室,广东汕头515041

出　　处：《汕头大学医学院学报》2010年第3期134-137,共4页Journal of Shantou University Medical College

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

摘　　要：目的:构建一系列细胞周期蛋白依赖激酶3(CDK3)在哺乳动物细胞的表达载体,并在真核细胞NIH3T3中进行初步表达。方法:从食管癌细胞中提取总RNA,RT-PCR扩增CDK3编码区,然后将PCR产物克隆到T载体;扩增后的CDK3片段分别亚克隆入pcDNA3-4、pNTAP和pEGFP等4种哺乳动物表达载体;将获得的表达载体转染小鼠成纤维细胞NIH3T3,并用蛋白免疫印迹法进行初步的CDK3表达分析。结果:PCR扩增获得约900 bp目的片段,经T载体克隆和DNA序列分析显示重组片段是人CDK3基因序列,全长915 bp;CDK3目的片段分别亚克隆入pcDNA3-4、pNTAP和pEGFP载体,获得相应表达载体;运用构建的表达载体,可以在真核细胞中表达出CDK3蛋白。结论:成功构建了CDK3系列哺乳动物细胞表达载体,并在NIH3T3中得到表达。Objective： To construct a series of cyclin-dependent kinase 3（CDK3）mammalian cell expression vectors, and to observe CDK3 expression in NIH3T3 cell. Methods： RNA was isolated from esophageal cancer cells and the full coding sequence of CDK3 gene was obtained by RT-PCR. The PCR product was then cloned into T vector and subsequently subcloned into four mammalian cell expression vectors（pcDNA3, peDNA4, pNTAP and pEGFP）. The expressing plasmids were transfected into NIH3T3 cells and the expression of CDK3 was detected by Western blot. Results： The PCR product was about 900 bp and the sequence analysis showed that it was the full coding sequenee of CDK3（915 bp）. The product was subeloned into the eukaryotic expression vectors, and four CDK.3 expression vectors were constructed. Results of Western blot showed that CDK3 could be expressed in the expression veetor-transfected cells. Conclusion： Four CDK3 mammalian cell expression vectors are successfully constructed and CDK3 is effectively expressed in NIH3T3 cells.

关 键 词：CDK3 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]