人谷胱甘肽硫转移酶M1基因的克隆及在大肠杆菌的温控表达  

Human liver glutathione-S-transferase M1 gene cloning and temperature-dependent expression in E.Coli

作　　者：陈萍萍[1] 吴逸明[2] 张朝武[1] 吴拥军[2] 

机构地区：[1]四川大学华西公共卫生学院,成都610041 [2]郑州大学公共卫生学院

出　　处：《卫生研究》2003年第3期236-239,共4页Journal of Hygiene Research

基　　金：河南省自然科学基金资助 (No .0 1 1 1 0 2 1 4 0 0 )

摘　　要：人谷胱甘肽硫转移酶M1基因的克隆及在大肠杆菌的温控高效表达 ,采用RT PCR技术从人肝脏组织总RNA中扩增谷胱甘肽硫转移酶M1基因的cDNA序列 ,将其插入到原核温控表达载体pBV2 2 0多克隆位点中 ,构建重组表达质粒 ,并用PCR扩增、酶切分析及序列测定等方法对重组质粒进行鉴定 ,并进行了温控表达 ,表达量约达到 2 8 3 %。人谷胱甘肽硫转移酶M1基因克隆到原核表达载体pBV2 2 0中 ,测序结果同Genbank中的人谷胱甘肽硫转移酶M基因序列比较 ,在 619位点C→A ,氨基酸由Pro→Thr,在 5 2 8位点C→T ,编码氨基酸仍为Asp。通过温控诱导 ,在 2 8kDa的表达量约达到 2 8 3 %。人谷胱甘肽硫转移酶M1原核温控表达载体pBV2 2 0的构建 ,为毒理学。In order to study the temperature-dependent expression of human liver glutathione-S-transferase M1 gene in prokaryotic cells, the recombinant of prokarytic expression vector pBV220 with human glutathione-S-transferase M1 gene was constructed. The recombined plasmid pBV220-hGSTM1 was verified with PCR, restriction analysis and sequencing. It's expression was induced with temperature-dependent in 42℃ and the expressed non-fusion protein in HD5α, with molecular weight of about 28kDa, was about 28.3% of the total cell protein determined by SDS-PAGE. The sequence of human liver glutathione-S-transferase M1 gene was verified to be correctly recombined with pBV220 compared with the same sequence in Gene Bank, and code 619 C→A, the amino acid changed from Pro to Thr was observed. The recombined plasmid pBV220-hGSTM1 may be applicable in toxicological and pharmacoeneticcal studies.

关 键 词：人谷胱甘肽硫转移酶M1 基因克隆 大肠杆菌 RT-PCR技术 

分 类 号：R394[医药卫生—医学遗传学]