人鼻咽癌相关基因NGX6在大肠杆菌中的诱导表达及纯化

Expression and Purification of NGX6 in Escherichia coli

NGX6是一个新克隆的鼻咽癌抑瘤基因候选者,为了进一步制备抗NGX6编码蛋白质的抗体和研究它的功能,本研究拟在原核表达系统中表达并纯化出NGX6蛋白.采用多聚酶链式反应(PCR)方法扩增出NGX6基因蛋白编码序列,构建该基因的融合表达质粒pGEX3X NGX6,导入大肠杆菌中,IPTG诱导表达,SDS PAGE电泳,Westernblot鉴定.用GST融合蛋白纯化试剂盒纯化融合蛋白.结果显示构建的融合表达质粒酶切和测序鉴定阅读框架正确,导入大肠杆菌中诱导后出现一条42kDa的新蛋白带,主要存在于细菌沉淀中,占总蛋白的26%,Westernblot鉴定其为GST NGX6融合蛋白,并进一步纯化出融合蛋白.研究表明NGX6基因编码蛋白质能够在原核细胞中表达,并能纯化出该种蛋白,为进一步深入研究该蛋白质的结构与功能打下了基础.

NGX6 is a novel Nasopharyngeal Carcinoma (NPC) putative suppressor gene.To prepare its antibody and further study it's function,the current study was designed to investigate the expression of NGX6 in Escherichia coli and its purification.The coding region of NGX6 was obtained by PCR,and a highlevel fusion protein expression vector pGEX3X/NGX6 was constructed by inserting fragment of the code region of NGX6 into a fusion protein expression vector pGEX3X.DNA sequencing and endonucleases digesting were used to check the coding region.The recombinant plasmid was transferred into E.coli.The NGX6/GST fusion protein was confirmed by Western blot.It was purified by GST purification kit.The result suggested that pGEX3X/NGX6 was constructed and proved that the coding region was correctly inserted into the vector.A new protein band of 42 kDa appeared on SDSPAGE after induction of IPTG.It amounted to 26% of the total protein.It existed mostly in precipitation of broken bacteria and was identified to be GST/NGX6 fusion protein.NGX6 can be expressed in Escherichia coli expression system and purified,which make it possible to do further investigation.