摘要
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 highlevel fusion protein expression vector pGEX3X/NGX6 was constructed by inserting fragment of the code region of NGX6 into a fusion protein expression vector pGEX3X.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 SDSPAGE 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.
出处
《生命科学研究》
CAS
CSCD
2003年第2期151-155,共5页
Life Science Research
基金
国家高技术"863"计划资助项目(102 01 05)
国家重点基础研究发展规划项目(973)(G1998051008)
国家自然科学基金资助项目(30000096)
国家自然科学基金资助项目(30271403)