翻译控制肿瘤蛋白TPT1的原核表达、纯化、抗体制备和初步应用  

Expression,purification,antibody preparation and identification of tumor protein translationally-controlled 1(TPT1)

作　　者：赵一璠[1] 李海芳[1] 汤石明[1] 王萌[1] 吴海东[1] 刘民[1] 李欣[1] 汤华[1] 

机构地区：[1]天津医科大学基础医学院生命科学中心实验室,天津300070

出　　处：《细胞与分子免疫学杂志》2010年第3期246-249,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30873017);天津市自然科学基金重点项目(08JCZDJC23300;09JCZDJC17500);国家高技术研究发展计划(863)资助项目(2007AA021808)

摘　　要：目的:原核表达并纯化翻译控制蛋白TPT1,免疫日本大耳白兔制备其抗体。方法:构建原核表达质粒pRSE-TA2-TPT1,转化大肠杆菌BL21(DE3),TPT1蛋白经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。融合蛋白通过Ni-NTA树脂亲和纯化后免疫兔子制备抗体血清。以间接ELISA法检测抗体效价,Western blot和免疫荧光染色鉴定抗体特异性。结果:在大肠埃希菌中诱导出高水平表达的TPT1融合蛋白,经亲和树脂纯化后免疫大白兔,获得了高特异性的抗TPT1抗血清。结论:成功构建原核表达质粒pRSETA2-TPT1,表达并纯化TPT1蛋白,制备出高滴度、高特异性的多克隆抗体.为进一步研究TPT1在肿瘤等疾病发生、发展过程及治疗中的作用奠定基础。AIM：To express and purify the fusion protein of TPT1（tumor protein translationally-controlled 1） in prokaryocytes and to prepare rabbit anti-TPT1 antibody.METHODS：The expression vector pRSETA2-TPT1 was reconstructed and transformed into BL21（DE3）.TPT1 fusion protein was induced by IPTG and the TPT1 fusion protein purified by Ni-NTA His Bind resin was used to immunize the rabbit.The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by Western blot and IF（immunofluorescence）.RESULTS：The TPT1 fusion protein was highly expressed in E.coli and specific polyclonal antibody was obtained after the immunization.CONCLUSION：The recombinant prokaryotic expression vector pRSETA2-TPT1 is successfully constructed and high expression of TPT1 is induced in E.coli,which may lay the basis for further study of the development and treatment of tumors and other diseases.

关 键 词：TPT1 原核表达 纯化 抗体 肿瘤 

分 类 号：R392.11[医药卫生—免疫学]