大肠杆菌偏嗜性人促甲状腺激素β亚基编码DNA序列的克隆及表达  

Cloning and Expression of DNA Sequence for Human Thyrotropin β Subunit Encoded By E.Coli Favorite Codes

作　　者：马香书[1] 马彦[1] 孙蓓[1] 王宝利[1] 赵学勤[1] 张镜宇[1] 郭刚[1] 

机构地区：[1]天津医科大学内分泌研究所,卫生部及天津市“激素与发育重点实验室”,300070

出　　处：《天津医药》2009年第5期340-342,共3页Tianjin Medical Journal

基　　金：天津市自然科学基金;“重中之重”学科建设专项基金(项目编号:05YFGDSF02700)

摘　　要：构建pGEX-3X/大肠杆菌偏嗜性人促甲状腺激素(hTSH)βDNA编码序列原核表达载体,并在E.coliDH5α中进行表达,用于GD(Graves diease)的实验研究。方法:用大肠杆菌偏嗜性密码子替换已知hTSHβ亚基基因编码序列中相应的密码子,按重新设计后的编码序列进行DNA化学合成,然后克隆入表达载体pGEX-3X并进行酶切电泳、测序鉴定,进而在E.coli DH5α中进行诱导表达。对所表达的GST融合蛋白进行SDS-PAGE、Western blotting及hTSH电化学发光免疫测定。结果:经鉴定,重组质粒pGEX-3X/hTSHβ含有与预期结果一致的435bp编码hTSHβ链的DNA序列。表达产物为hTSHβ与GST融合蛋白,分子质量约42ku,于SDS-PAGE中的相应部位可见深染的特异性蛋白条带,可被GST抗体识别并可被抗hTSH抗体检测。结论:实验成功建立了hTSHβ肽链的体外表达系统,为进一步制备hTSHAb及进行GD的研究奠定了基础。Objective：To obtain E.coli favorite DNA sequence encoding human thyrotropin β subunit and transform the cloned DNA into E.coli DH5et for stable expression. Methods：The human thyrotropin β subunit encoding sequence of DNA was modified according to the known E.coil favorite codes and was synthesized chemically.The synthesized DNA sequence was cloned into plasmid pGEX-3X and the recombinant plasmid was identified with restriction mapping and DNA sequencing. Plasmid pGEX-3X/hTSHβ was then transformed into E.coli l）H5α and the host cell was induced to express GST fusion protein by IPTG. Western blotting was performed with rabbit anti-GST IgG as primary antibody and goat anti-rabbit IgG HRP as secondary antibody.The GST fusion protein was also detected by Electrochemiluminescence immunoassay （ECLI）. ReSults ：The restriction mapping and DNA sequencing confirmed that pGEX-3X/hTSHβ had been constructed successfully. The recombinant plasmid could stably express the fusion protein GST-hTSHβ mainly in the form of insoluble inclusion bodies from E.coil. Conclusion：The prokaryotic expression system of hTSH13 was constructed successfully, which will contribute to the studies on Graves＇ disease and as an immunogen used for antibody preparation. Key words thyrotropin eseherichia coli eodon cloning, organism gene expression

关 键 词：促甲状腺素 大肠杆菌 密码子 克隆 生物 基因表达 

分 类 号：R373.9[医药卫生—病原生物学] R446.61[医药卫生—基础医学]