人甲状腺刺激激素受体cDNA的克隆及真核表达载体的构建.pdfVIP

新乡医学院学报 Journal of Xinxiang Medical College Vo1．25 No．1 Jan． 2008 ·1· · 基础研究 · 人甲状腺刺激激素受体cDNA的克隆及真核表达载体的构建 李晓永，冯绮文，董 艳，周亚芹，葛勤敏，张洪梅，苏 青 (上海交通大学医学院新华医院内分泌科，上海 200092) 摘要： 目的 克隆人甲状腺刺激激素受体(hTSHR)基因cDNA并构建其真核表达载体。方法 以人甲状腺组织 cDNA为模板，聚合酶链反应(PCR)扩增hTSHR基因编码区的全部序列，克隆人pGEM—T载体中，经限制性内切酶、DNA 序列分析鉴定目的基因后，定向亚克隆到真核细胞表达载体pcDNA3．1中，并进行双酶切鉴定。结果 PCR扩增的特 异性片段长度为2 337 bp，以此构建的pGEM．T．hTSHR克隆载体，经限制性内切酶酶切及DNA序列分析证实载体中带 有hTSHR基因编码区的目的片段，重组质粒pcDNA3．1-hTSHR经 n I和Xba I双酶切后显示5 1000 bp和2 300 bp左 右的2个条带，DNA序列分析显示与GenBank中的hTSHR基因cDNA序列一致，证明hTSHR基因已成功克隆人真核细 胞表达载体pcDNA3．1中。结论 成功构建了野生型pcDNA3．1-hTSHR重组真核表达载体。 关键词： 甲状腺刺激激素受体；克隆；真核表达载体 中图分类号：R78 文献标识码：A 文章编号：1004-7239(2008)01-0001—04 Cloning of human thyroid stimulating hormone receptor gene and construction of its eukaryotic ex- pression vector LI Xiao—yong，FENG Qi-wen，DONG Yan，et al (Department ofEndocrinology，Xinhua Hospital，School ofMedwine，Shanghai Jiaotong University，Shanghai 200092，China) Abstract：Objective To clone human thyroid stimulating hormone receptor(hTSHR)gene and construct its eukaryotic expression vector．Methods The whole coding sequence of hTSHR gene was amplified by polymerase chain reaction(PCR) applied with human thyroid cDNA．The fragment Was inserted into cloning vector pGEM—T and the recombinant pGEM—T—hTSHR was identified by double digestion with restriction enzymes and sequencing．The hTSHR cDNA fragment was subcloned into pcD— NA3．1 plasmid．The recombinant pcDNA3．1一hTSHR was identified by double digestion with restriction enzymes and sequen— cing．Results Th e length of the specific fragment by PCR Was 2337bp，and the pGEM—T—hTSHR plasmid Was identified by the salne way．The sequence was identical to that of hTSHR cDNA in GenBank．Th e recombinant pcDNA3．1一hTSHR plasmid Was separated into two bands，approximately 5 100 kb and 2300bp，by using respective restriction enzymes Kpn I and Xba I，and the sequence Was identical to that of hTSHR cDNA in GenBank，sugge