Tec激酶区作用蛋白RAI16的原核表达和纯化  

Prokaryotic expression and purification of retinoic acid induced 16 interacting with Tec kinase domain

作　　者：许文[1] 王阁[1] 邓婧[1] 杨进[1] 郑继军[1] 王红中[1] 胡庆[1] 王东[1] 李增鹏[1] 杨志祥[1] 

机构地区：[1]中国人民解放军第三军医大学大坪医院野战外科研究所肿瘤中心,重庆市400042

出　　处：《世界华人消化杂志》2008年第12期1350-1354,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30370341;No.30570410;全国优秀博士专项基金资助项目;No.200261~~

摘　　要：目的:利用原核表达系统构建Tec激酶区作用蛋白RAI16的融合蛋白表达载体,并进行表达条件的优化和初步纯化.方法:设计基因拼接引物,合成RAI16cDNA序列,将其连接于克隆载体pMD18-T中;将酶切、纯化的RAI16基因与pGEX4T-2载体相连接、转化、筛选.将鉴定阳性的重组子质粒转人大肠杆菌BL-21表达菌中,采用SDS-PAGE电泳分析不同浓度诱导剂异丙基硫代-D-半乳糖苷(IPTG)、不同诱导温度、不同诱导时间下目的蛋白的表达.结果:成功构建RAI16蛋白原核表达载体.采用0.4mmol/LIPTG、30℃诱导4h,获得较高表达目的蛋白.融合蛋白主要以包涵体形式表达,将包涵体进行尿素法变性复性和亲和层析柱处理后,获得可溶的高纯度GST-多肽融合蛋白.结论:Tec激酶区作用蛋白RAI16融合蛋白表达的载体和纯化,是制备RAI16多抗以及进一步验证与Tec体外结合作用实验的基础.AIM： To construct and purify an active region of prokaryotic expression vector retinoic acid induced 16 （RAI16） interacting with Tec kinase domain. METHODS： TRAI16 cDNA sequence was synthesized, and then linked to pMD18-T vector. After enzyme digestion, the purified target fragment was linked to the expression vector pGEX4T-2, which was then transferred and screened. After the positive recombinants were transferred into human E.coil BL-21, the expression was induced by different concentrations of isopropyl β-D-thiogalactoside （IPTG） at different temperatures and culture time periods. The expression products were analyzed by SDS-PAGE. RESULTS： RAI16 cDNA was successfully cloned in pGEX4T-2 plasmid. Using 0.4 mmol/L IPTG at 30℃ for 4 h, the soluble target protein was expressed efficiently. SDS-PAGE revealed glutathione S-transferase-RAI16 fusion protein bands expressed mainly in the form of inclusion bodies. CONCLUSION： High expression of the extracellular region of Tec fusion protein is attained using E.coil BL-21, and the soluble target protein without any additional amino acid is successfully purified.

关 键 词：Tec蛋白 视黄酸诱导蛋白16 核苷酸 蛋白质结构 

分 类 号：R996.3[医药卫生—毒理学] R394[医药卫生—药学]