人CDK7基因真核表达载体的构建与鉴定  

Construction and identification of plasmid vector of CDK7

作　　者：冀全博[1,2] 徐小洁[1] 张强[2] 梁迎春[1] 王涛[1] 李玲[1] 黄蓉[1] 周丽英[1] 史平安[1] 王岩[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]解放军总医院骨科,北京100853

出　　处：《军事医学》2014年第12期932-935,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31100604;81101387);北京市自然科学基金资助项目(7132155)

摘　　要：目的构建带Myc标签的细胞周期蛋白依赖性激酶7(cyclin-dependent kinase 7,CDK7)的真核表达载体,获得其表达产物,并验证该激酶与已知相互作用蛋白P53之间的相互作用。方法应用PCR技术从人乳腺文库中扩增出CDK7全长编码区基因,将其克隆到p XJ-40载体中;继而重组质粒转染人胚肾293T细胞,以SDS-PAGE和Western印迹鉴定表达情况;免疫共沉淀检测Myc-CDK7与FLAG-p53的相互作用。结果双酶切和基因测序鉴定显示,Myc-CDK7真核表达质粒克隆构建成功;SDS-PAGE和Western印迹结果表明,Myc-CDK7转染人胚肾293T细胞后成功表达;免疫共沉淀显示,重组Myc-CDK7与已知相互作用蛋白P53在蛋白质水平上具有相互作用,证实其具有生物学活性。结论成功构建Myc-CDK7的真核表达载体,为进一步探讨Myc-CDK7对细胞周期的调控奠定了实验基础。Objective To construct the eukaryotic expression vector of cyclin-dependent kinase（CDK） 7 labeled with Myc tag,obtain the expressed product,and identify its interaction with FLAG-P53 at the protein level.Methods Human CDK7 coding gene region amplified from the mammary c DNA library by PCR was inserted into the p XJ-40 vector.The recombinant plasmid Myc-CDK7 transfected into human 293 T cell lines was investigated and examined by SDS-PAGE and Western blotting.In addition,assay was applied to determine the interaction between Myc-CDK7 and FLAG-p53.Results The coding region of CDK7 was successfully amplified by PCR and cloned into p XJ-40 vector,which was identified by double enzyme digestion and gene sequencing.Myc-CDK7 was successfully expressed in human 293 T cell lines according to SDS-PAGE and Western blotting assay indicated that Myc-CDK7 could interact with P53 protein,which verified its known function.Conclusion The eukaryotic expression vector Myc-CDK7 is successfully obtained,which will contribute to further research on CDK7-mediated cell cycle regulation.

关 键 词：细胞周期蛋白质依赖激酶类 基因 p53 真核表达 

分 类 号：Q786[生物学—分子生物学]