重组人甲状旁腺激素相关蛋白1-141的表达和生物活性测定  

Cloning and Expression of Recombinant Human Parathyroid Hormone Related Protein 1-141

作　　者：刘新宇[1] 梁东春[2] 左爱军[2] 张镜宇[2] 郭刚[2] 

机构地区：[1]天津医科大学生物化学教研室,300070 [2]天津市内分泌研究所

出　　处：《天津医药》2007年第1期33-36,共4页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(项目编号:39970348);天津市自然科学基金重点项目(项目编号:993804211)

摘　　要：目的:建立表达及纯化重组人甲状旁腺激素相关蛋白1-141(rhPTHrP1-141)的方法。方法:将已构建好的表达载体pQE-30Xa/hPTHrP1-141重组质粒转化大肠杆菌M15[PREP4],IPTG诱导表达,WesternBlot鉴定表达产物。金属螯合亲和层析去除杂蛋白后,超滤离心进一步纯化目的蛋白。使用UMR106细胞测定所制备的rhPTHrP1-141对cAMP生成的刺激作用。结果:SDS-PAGE可见与预期大小相符的蛋白条带,WesternBlot证实该条带即为rhPTHrP1-141。目的蛋白经纯化后,能刺激UMR106细胞内cAMP浓度升高,有剂量依赖关系。结论:成功建立了rhPTHrP1-141的制备及纯化方法,PTHrP1-141的表达量可达到60mg/L培养基。表达产物经体外实验证实具有生物活性。Objective： To express and purify of recombinant human parathyroid hormone related protein 1-141. Methods： The constructed expression vector pQE-30Xa/hPTHrP1-141 recombinant plasmid was transformed into E.coli M15 [PREP4]. Then the transforming was induced to express by increasing the concentration of IPTG. Expressing product was verified by Western blot, hybridprotein was removed by metal-chelating affinity chromatograph,finaUy, interest protein was further purified by ultrafihration centrifugalization and rhPTHrP1-141 was assayed by UMR106 to see whether the concentration of cAMP was stimulated to increase or not. Results： The anticipated protein strap that was confirmed to be rhPTHrP1-141 by Western blot was appeared by SDS-PAGE. Purified targeted protein was able to stimulate UMR106 cell to increase its intracellular cAMP, furthermore the result was dose-dependent. Conclusion： Recombinant human parathyroid hormone related protein 1-141 was expressed and purified successfully and its expression quatity density can achieve to the 60 mg/L medium. Expression products have biological activity in vitro.

关 键 词：甲状旁腺激素相关蛋白质 重组蛋白质类 大肠杆菌 基因表达调控 

分 类 号：R965.3[医药卫生—药理学]