大鼠BTCe蛋白原核表达及功能鉴定  

Expression and Purification of Rat EGF-like Domain of BTCe

作　　者：李鸿[1] 周伟斌[1] 顾燕云[1] 周文中[1] 张迪[1] 骆天红[1] 李果[1] 罗敏[1] 

机构地区：[1]上海交通大学医学院瑞金医院上海市内分泌代谢病研究所,上海200025

出　　处：《上海交通大学学报（医学版）》2006年第1期51-53,共3页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家重点基础研究发展规划("973"项目)(2002BA711A05);国家自然科学基金(30470817);上海市教委重点学科基金(Y0204)资助项目

摘　　要：目的原核表达获得大量具有生物活性的大鼠BTCe蛋白。方法RT-PCR法从大鼠肾脏中扩增出150bp BTCe片段,插入原核表达载体pGEX-4T-2,构建质粒pGEX-BTCe。转化BL-21菌,IPTG低温诱导表达融合蛋白,亲和层析法纯化获得融合蛋白,凝血酶酶切后获得目的蛋白,经SDS-PAGE、Western blot及质谱鉴定。以0.5nmol/L BTCe作用于3T3-L1细胞连续培养4d,MTT法检测其促增殖能力。结果融合蛋白以水溶性形式分泌于大肠杆菌BL-21胞浆中。酶切后的目的蛋白经质谱证实其与BTCe蛋白氨基酸序列相符;作用于3T3-L1细胞可明显促进其增殖。结论大鼠BTCe蛋白在pGEX-4T-2原核表达载体可获得高效表达,所纯化蛋白具有促进3T3-L1细胞体外增殖的作用。Objective To obtain abundant rat EGF-like domain of betacellulin （ BTCe ） with biological activity. Methods A 150 bp rat BTCe coding was amplified by RT-PCR from the rat kidney. The fragment was inserted into vector pGEX4T-2 to construct recombinant plasmid pGEX-BTCe. Following transformation into E. coli BL-21, the fusion protein was expressed under IPTG induction. After cleavage reaction by thrombin protease, BTCe was obtained from fusion protein. SDS-PAGE, Western blot and mass spectrometry （MS） were used to determine the expression and purification of the expected protein. 0.5 nmol/L BTCe was added to culture 3T3-L1 cells to study biological activity. Results Lots of fusion protein were produced and the BTCe protein was confirmed by MS. The purified BTCe protein can stimulate the proliferation of 3T3-L1 cells similar to BTC protein. Conclusion Rat BTCe protein can be successfully obtained from the pGEX4T-2 system with biological activity of stimulating the proliferation of 3T3-L1 cells.

关 键 词：BTCe蛋白 克隆 融合蛋白 

分 类 号：R346[医药卫生—基础医学]