胶质纤维酸性蛋白的原核表达、纯化及鉴定  

Expression, Purification and Identification of Glial Fibillary Acidic Protein

作　　者：吕立权[1] 曹鹏[1] 胡国汉[1] 骆纯[1] 侯立军[1] 卢亦成[1] 

机构地区：[1]上海长征医院神经外科,上海200003

出　　处：《中国临床神经外科杂志》2009年第10期606-609,共4页Chinese Journal of Clinical Neurosurgery

基　　金：国家自然科学基金青年基金项目(30600635)

摘　　要：目的为获得高纯度的人胶质纤维酸性蛋白(GFAP)。方法从人脑胶质瘤组织中提取mRNA,采用RT-PCR的方法克隆GFAP基因全长序列,利用限制性内切酶EcoRⅠ和XhoⅠ将GFAP基因插入到原核表达载体pGEX4T2,对重组表达载体pGEX4T2-GFAP进行双酶切和测序鉴定。选择测序正确的质粒转化大肠杆菌BL21,用终浓度为1mmol/L的异丙基-β-D-硫酸代半乳糖苷(IPTG)进行诱导表达GFAP-GST融合蛋白。经亲和层析纯化获得GFAP融合蛋白,并通过SDS-PAGE和western blot进行鉴定。结果重组质粒经双酶切和测序鉴定证实插入序列准确无误。宿主菌BL21经IPTG诱导表达并纯化得到目的蛋白GFAP-GST融合蛋白,经SDS-PAGE鉴定分子量约为70～90kD,符合预期,westernblot证实该目的蛋白能被特异性抗人GFAP多克隆抗体识别。结论成功构建GFAP表达载体,并可通过原核表达获得高纯度的目的蛋白,为进一步开展相关研究打下基础。Objective To gain highly pure human glial fibillary acidic protein（GFAP）. Methods The mRNA isolated from human brain glioma tissues was subjected to RT-PCR to obtain full lenghth GFAP gene, which was inserted into prokaryotic vector PGEX4T2 with restriction enzymes EcoR Ⅰ and Xho Ⅰ. Recombinant expression plasmid PGEX4T2-GFAP was confirmed by restriction endonuclease mapping and sequencing. The interesting plasmid PGEX4T2-GFAP was transformed into E.Coli host BL21 and induced by 1 mmol/L isopropyl β-D-1-thiogalactopyranoside （IPTG）. The GFAP was purified by SP and Mono Q chromatography, and characterized by SDS-PAGE and western blotting. Results The GFAP gene was inserted into vector PGEX4T2 accurately. GFAP expressed by E.Coli host BL21 was gained by IPTG induction and purification. SDS-PAGE revealed a new protein with from 70 to 90 kD band. The multiclonal antibodies against GFAP reacted strongly with the interest protein. Conclusion That the prokaryotic expression vector of GFAP was successfully constructed may provide a basis for further relative studies.

关 键 词：胶质纤维酸性蛋白 原核表达载体 表达 

分 类 号：R392.9[医药卫生—免疫学]