人胚生殖嵴干细胞多能性调节基因Oct4的克隆及其在大肠杆菌中的表达(英文)  

Cloning of human embryonic germ cell pluripotency regulation gene Oct4 and its expression in E.coli

作　　者：刘善荣[1] 王庆敏[2] 杨玲[1] 汤淑萍[1] 惠宁[3] 蒋正[3] 戚中田[4] 刘厚奇[1] 

机构地区：[1]第二军医大学基础医学部组织胚胎学教研室,上海200433 [2]第二军医大学基础医学部医学遗传学教研室 [3]第二军医大学长海医院妇产科 [4]第二军医大学基础医学部微生物学教研室

出　　处：《第二军医大学学报》2003年第9期937-940,共4页Academic Journal of Second Military Medical University

基　　金：NationalFoundationforOutstandingYoungSci entist(No .3 982 5 116) ;CooperationFoundationforoverseasYoungSci entist(No .3 992 80 0 9)

摘　　要：目的 :克隆人胚生殖系细胞多能性调节基因Oct4 ,并使其在大肠杆菌中表达。 方法 :取 4～ 6周人胚胎分离生殖嵴和背侧肠系膜 ,利用RT PCR技术扩增编码Oct4的全长cDNA ,并插入到原核表达载体 pGEX5T中 ,构建成 pGEX5T Oct4重组质粒。β硫代半乳糖苷 (IPTG)诱导使该基因在k80 2菌中表达。 结果 :用PCR方法扩增获得大小约 1 .1kb条带 ;全自动测序与GenBank中登录的序列 97%同源。获得了pGEX5T Oct4重组子并在k80 2菌中获得了表达 ,SDS PAGE分析有一特异性的 6 2 0 0 0条带。 结论 :扩增和克隆了人胚生殖系细胞多能性调节蛋白Oct4全长cDNA并在大肠杆菌中获得表达 。Objective: To clone the gene encoding Oct4 protein regulating human embryonic germ cell pluripotency and to express it in E.coli (k802). Methods: Total RNA was extracted from gonadal ridges and mesenteries of 4 to 6 week old human embryos and cDNA amplification was performed by Reverse Transcription Polymerase Chain Reaction (RT PCR). The full length Oct4 cDNA was cloned into prokaryotic expressing vector pGEX5T and the product was sequenced. PGEXT oct4 was then expressed in k802 E.coli . Results: One specific band of 1.1 kb was obtained and had 97% affinity to that reported in GenBank. The protein was successfully expressed,exhibiting a 62 000 band by SDS PAGE analysis. Conclusion: The full length Oct4 cDNA has been amplified and cloned,and Oct4 protein is expressed successfully in k802 E.coli . This may be helpful in future studies on pluripotency and differentiation of human embryonic germ cells.

关 键 词：人胚生殖嵴干细胞 多能性调节基因 OCT4 克隆 基因克隆 基因表达 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学] R321[医药卫生—基础医学]