人干细胞因子全长cDNA的基因扩增与克隆  

Amplification and cloning of human stem cell factor full-length cDNA by RT-PCR

作　　者：谭运年[1] 谭文斌[2] 彭兴华[2] 

机构地区：[1]广东医学院附属医院妇产科研究室,广东湛江524001 [2]湖南医科大学分子生物学研究中心,长沙410078

出　　处：《广东医学》2001年第1期16-18,共3页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(项目编号:39700050)

摘　　要：目的 探讨人干细胞因子的结构与功能及其在真核细胞中的表达与调控的机制，首先克隆了人干细胞因子金长ｃＤＮＡ。方法 用ＲＰＭ１６４０，体积分数为１０％的小牛血清培养比闷细胞，抽提ＲＮＡ，行ＲＴ－ＰＣＢ，纯化ＰＣＲ产物，与ｐＧＥＭ－Ｔ克隆载体连接，转化，铺板，筛选阳性克隆，酶切鉴定并进行序列测定。结果 通过酶切鉴定并进行序列测定，成功地获得了人干细胞固子全长ｃＤＮＡ的基因克隆。结论 人干细胞因子全长ｃＤＮＡ的基固克隆的构建成功，为其结构与功能的研究及其在真核细胞中的表达与调控的研究提供了一定的物质基础。Objective To study the expression and regulation of human stem cell factor in eukaryotic cell, its full - length cD-NA was amplified by RT - PCR and its cloning vector was constructed. Methods HepG2 cells were cultured in RPMI1640 containing 10% bovine serum, and the cultured cells were harvested and their RNA was extracted;The 1.14 kb cDNA was amplified by RT - PCR, and the full - length cDNA fragment was inserted in the pGEM - T vector. Results The recombinant plasmid was cleaved with restrictive endonuclease and sequencing result showed that the cloning vector was successfully created. Conclusion The recombinant construct of the full - length cDNA provids available conditions for further study on expression and regulation of stem cell factor.

关 键 词：干细胞因子 脱氧核糖核酸 克隆 基因扩增 CDNA 

分 类 号：R331.2[医药卫生—人体生理学] R394[医药卫生—基础医学]