RUNX3全长和不同结构域原核表达载体的构建及其重组蛋白表达  

Construction of prokaryotic expression vector of overall length and different domains of RUNX3and expressions of their recombinant proteins

作　　者：宋艳艳[1] 王桂玲[1] 

机构地区：[1]中国医科大学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：《吉林大学学报（医学版）》2013年第6期1112-1115,共4页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30871294);辽宁省自然科学基金资助课题(201102277)

摘　　要：目的:构建RUNX3全长和不同结构域的原核表达载体并表达其重组蛋白。方法:以pcDNA3.1-myc-RUNX3为模板,采用PCR法扩增RUNX3全长和各个结构域片段,通过EcoRⅠ和BamHⅠ酶切位点将RUNX3全长和不同结构域定向插入谷胱甘肽转移酶(GST)融合表达载体pGEX-4T-2中,在大肠杆菌(E.coli)BL21中诱导其融合蛋白表达,SDS-PAGE电泳检测其蛋白表达情况。结果:EcoRⅠ和BamHⅠ双酶切后得到与预期大小相符的FL(1 245bp)、ΔRunt(843bp)、Nter(159bp)、Runt(402bp)和Cter(684bp)5个载体片段。SDS-PAGE电泳检测,RUNX3全长及各个结构域截短GST-RUNX3截短融合蛋白(GST-FL、GST-Runt、GST-Nter和GST-Cter)相对分子质量分别为72 000、40 000、32 000和51 000。结论:成功构建RUNX3全长和各个结构域截短的GST标签的原核表达载体,并实现其重组蛋白在原核细胞中的表达。Objective To construct the prokaryotic expression vectors of overall length and different domains of RUNX3 and to identify the expressions of their recombinant proteins. Methods The coding sequence of overall length and different domain truncations of RUNX3 were amplified from the pcDNA3.1-myc-RUNX3 plasmid by PCR method and inserted into glutathione transferase （（;-ST） fusion expression vector pGEX 4T 2 through EcoR I and BamH I restriction enzyme sites. Then they were transformed into E. coli BL21 and the fusion proteins were induced and identified by SD-PAGE electrophoresis. Results The vector fragments FL （1 245 bp）, ＆Runt （843 bp）, Nter （159 bp）, Runt （402 bp）, and Cter （684 bp） which were consistent with the expected fragments were obtained after double enzyme digestion of EcoR I and BarnH I . The SDS PAGE electrophoresis result showed that the relative molecular mass of GST RUNX3 truncated fusion proteins of overall length and different domains of RUNX3 （GSTFL, GST-Runt, GST-Nter and GST-Cter） were 72 000, 40 000, 32 000, and 51 000, respectively. Conclusion GST tagged prokaryotic expression vectors overall length and different truncated regions of RUNX3 are constructed and their recombinant proteins are expressed successfully.

关 键 词：RUNX3 载体 蛋白诱导 蛋白表达 

分 类 号：Q257[生物学—细胞生物学]