人GSTM1TV2基因的克隆及温控表达

Temperature-dependent expression and construction of prokarytic expression vector pBV220-GSTM1TV2 gene from human lung cDNA library and partial sequence determination

摘要:
目的克隆人GSTM1TV2基因并在大肠杆菌的温控高效表达。
方法用RT-PCR技术从人肺组织总RNA中分离扩增人GSTM1TV2基因的cDNA序列, 将其插入到原核温控表达载体pBV220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。
结果人GSTM1TV2克隆到原核表达载体pBV220中, 测序结果同Genbank序列比较, 完全一致。通过温控诱导, 在26kDa的表达量约达到33%。
结论 pBV220-GSTM1TV2原核温控表达载体的构建, 为毒理学、遗传药理学的研究提供应用基础。

Abstract:
Objective To express on temperature-dependent and constructan prokaryotic expression vector carrying human GSTM1TV2 gene and expression with high efficiency in E.coli DH5a.
Methods The GSTM1TV2 cDNA was amplified from human lung total RNAs by RT-PCR and was recombined with prokarytic expression vector pBV220.The recombined plasmid pBV220-GSTM1TV2 were verified with PCR, restriction analysis and sequencing determination and induced with temperature-depenent in 42℃.
Results The expressed non-fusion protein, with molecular weight of about 26 kDa, was about 33% of the total cell protein by SDS-PAGE. Human GSTM1TV2 was recombined correctly with pBV220.Comparing with Genbank, it is correct.
Conclusion The recombined plasmid pBV220-GSTM1TV2 was constructed for research on toxicology and pharmacogenetics.