组蛋白脱乙酰化酶GST-HDAC1融合蛋白载体的构建及其在大肠杆菌中的表达  

Construction of GST-HDAC1 fusion protein vector and its expression in E.coli

作　　者：王桂玲[1] 陈薇[1] 王孝会[1] 姜佩佳[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,沈阳110001

出　　处：《解剖科学进展》2011年第6期535-537,共3页Progress of Anatomical Sciences

基　　金：国家自然科学基金资助项目(No.30871294);辽宁省自然科学基金资助项目(No.201102277)

摘　　要：目的构建GST-HDAC1融合蛋白表达载体,并在大肠埃希菌(E.coli)中诱导表达。方法以质粒pcDNA3.1-HDAC1为模板进行PCR,通过EcoR1单酶切位点将HDAC1插入pGEX-5X-1中,构建原核表达质粒pGEX-5X-1-HDAC1,并转化E.coli DH5α,通过利用载体上的BamH1酶切位点和HDAC1上Nde1酶切位点来筛选阳性重组子,DNA序列测定正确后,转入化E.coli BL21中,异丙基硫代β-D半乳糖苷诱导表达,鉴定。结果酶切后获得940bp片段为正向;如获得500bp片段为反向。重组质粒测序后与GenBank进行同源性比对证明导入片段为HDAC1,成功构建了原核表达质粒pGEX-5X-1-HDAC1,并用SDS-PAGE方法证实了GST-HDAC1融合蛋白在大肠杆菌中的表达。结论成功构建了HDAC1原核表达载体,并证实了融合蛋白在大肠埃希菌中的表达,为进一步纯化HDAC1蛋白及与其它蛋白的关系奠定了基础。Objective To construct GST-HDAC1 fusion protein expression vector and induce its expression in Escherichia coli（）.Methods The coding sequence of HDAC1 was amplified from the plasmid pcDNA3.1-HDAC1 by PCR and inserted into pGEX-5X-1 by EcoR1.The positive recombinants were identified by restriction endonuclease digestion and DNA sequencing.Then they were transformed into BL21,induced by IPTG and identified by SDS PAGE.Results The length of the forward fragment was 950bp identified by enzymes digestion and the length of reverse fragment was 500bp.The sequence was compared with that in GenBank for homology and the improved prokaryotic expression plasmid pGEX-5X-1-HDAC1 was successfully constructed.The desired GST-HDAC1 fusion protein was expressed in by Western blot.Conclusion The prokaryotic expression plasmid of HDAC1 was successfully constructed and the fusion proteins was expressed in,which provides the basis for the further research on purifying HDAC1 protein and the relationship with other protein.

关 键 词：GST-HDAC1融合蛋白 原核表达 

分 类 号：Q26[生物学—细胞生物学]