原核表达载体GST-RUNX3的构建及其在大肠杆菌表达  

Construction and expression of prokaryotic expression vector GST-RUNX3 in E.coli

作　　者：王桂玲[1] 王孝会[1] 都田赵[1] 陈薇[1] 姜佩家[1] 

机构地区：[1]中国医科大学基础医学院细胞生物学教研室卫生部细胞生物学重点实验室,辽宁沈阳110001

出　　处：《解剖科学进展》2011年第4期375-377,共3页Progress of Anatomical Sciences

基　　金：国家自然科学基金资助项目(No.30871294)

摘　　要：目的构建GST/RUNX3融合蛋白表达载体,并在大肠埃希菌(E.coli)中诱导表达。方法利用Kpn1和Xho1位点酶切pcDNA3.1-RUNX3质粒,将获得的1300bpRUNX3编码序列插入到pGEX-4T-2中,构建pGEX-4T-2-RUNX3质粒,并转化E.coli DH5α,筛选阳性重组子,酶切鉴定和DNA序列测定正确后,转入BL21中,经IPTG诱导表达后SDS-PAGE电泳鉴定。结果酶切pGEX-4T-2-RUNX3质粒获得1300bpRUNX3片段,经测序后获得的DNA序列与GenBank进行同源性比对,证实pGEX-4T-2-RUNX3构建成功。在E.coli BL21用IPTG进行诱导表达后,SDS-PAGE电泳方法证实了GST-RUNX3融合蛋白表达。结论成功构建了RUNX3原核表达载体,其融合蛋白在大肠埃希菌表达,为进一步纯化RUNX3蛋白和研究RUNX3的生物学功能奠定了基础。Objective To construct GST/RUNX3 fusion protein expression vector and induce its expression in Escherichia coli（E.coli）.Methods The coding sequence of RUNX3 containing 1300bp was obtained from the pcDNA3.1-RUNX3 plasmid by KpnI and Xho1 and inserted into pGEX-4T-2 vector.The positive recombinants were transformed to E.coli DH5α and screened by restriction endonuclease.The identified pGEX-4T-2-RUNX3 plasmid by sequencing was expressed in E.coli BL21 by IPTG induction and confirmed by SDS-PAGE.Results The coding 1300bp sequence of RUNX3 was obtained by digestion of the pGEX-4T-2-RUNX3 plasmid with KpnI and Xho1,and was homologous with the sequence of RUNX3 in GenBank,which indicates that the pGEX 4T-2-RUNX3 plasmid was successfully constructed.The GST-RUNX3 fusion protein was confirmed by SDS-PAGE to be expressed in E.coli.Conclusion The successful construction of prokaryotic expression plasmid of RUNX3 provides the basis for further research on purifying RUNX3 protein and the biological function of RUNX3

关 键 词：pGEX-4T-2-RUNX3质粒 原核表达 融合蛋白 大肠埃希菌 

分 类 号：R394[医药卫生—医学遗传学]