G156A六氧甲基鸟嘌呤-DNA-甲基转移酶原核表达载体的构建和表达

The Construction and Expression of Prokaryotic Expression Vector of G156A O^6-methylguanine-DNA-methyltransferase

目的 :构建原核表达载体pPROEXHTb G15 6AMGMT ,观察突变型G15 6AMGMT在大肠杆菌中的表达情况。方法 :通过基因重组技术将G15 6AMGMT亚克隆至pPROEXHTb原核表达载体 ,在大肠杆菌DH5α中经异丙基硫代 β D半乳糖苷 (IPTG)诱导表达。 结果 :经PCR及酶切分析证实 ,成功构建了pPROEXHTb G15 6AMGMT原核表达载体 ,经SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)显示重组克隆化基因pPROEXHTb G15 6AMGMT在大肠杆菌DH5α中有表达。结论 :G15 6AMGMT在大肠杆菌DH5α中有表达 ,为获取突变型MGMT工程蛋白奠定了基础。

Objective: To construct recombinant prokaryotic expression vector pPROEX HTb-G156A MGMT and to observe its expression in E.coli DH5α. Methods: The G156 MGMT cDNA was subcloned into pPROEX HTb. G156A MGMT protein was induced by IPTG to express in E.coli. Results: The recombinant vector, pPROEX HTb-G156A MGMT, was successfully constructed and confirmed by the restriction endonuclease and PCR analysis. G156A MGMT protein expressed successflly in E.coli. Conclusions: G156A MGMT expresses in E.coli under the induction of IPTG. The study provides a foundation to the research aimed at obtaining mutant protein of G156A MGMT.