TAT-MafA融合蛋白诱导小肠细胞系IEC-6表达胰岛素  

TAT-MafA Inducing Intestinal Epithelial Cells IEC-6 into Insulin Positive Cells

作　　者：罗海兰[1] 毋慧玲[2] 路君[2] 卢大儒[2] 沈坤堂[3] 金坚[1] 

机构地区：[1]江南大学生物工程学院,无锡214122 [2]复旦大学遗传工程国家重点实验室,上海200433 [3]复旦大学附属中山医院,上海200032

出　　处：《生物技术通报》2008年第5期140-144,共5页Biotechnology Bulletin

基　　金：上海市科委资助项目(05ZR14028)

摘　　要：借助蛋白质转导技术,利用胰岛β细胞转录因子MafA诱导小肠细胞系IEC-6表达胰岛素,为糖尿病的治疗提供新的方法。将TAT基因与MafA基因一起插入p32a(+)载体中,构建TAT-MafA融合蛋白原核表达载体,转化BL21获得表达菌株,经IPTG诱导和Ni柱亲和层析纯化TAT-MafA融合蛋白,用1μM浓度的该融合蛋白孵育IEC-6细胞12h和24h后,分别进行进胞检测和胰岛素表达检测。实验结果表明,细胞免疫荧光和Western-blot方法检测到经TAT-MafA融合蛋白作用12h后的IEC-6细胞中有该蛋白,并经细胞免疫荧光和RT-PCR的方法检测出TAT-MafA作用24h后的IEC-6细胞中有胰岛素的表达。因此,TAT-MafA融合蛋白可以高效进入小肠细胞系IEC-6细胞,并且进胞后仍保持MafA原有的生物学活性,能启动胰岛素基因的表达,诱导IEC-6成为胰岛素表达细胞。The aim was to use transcription factor MafA to induce intestinal epithelial cells IEC-6 into insulin positive cells by protein transduction technology. Prokaryotic expression vectors of TAT-MafA fusion protein was constructed by inserting genes encode TAT and MafA into p32a （＋）vector,and the fusion protein was obtained,after incubating IEC-6 with 1 p.M TAT-MafA for 12h and 24h.The internalization of the protein was investtigated by using immunostaining and western blot analysis,and insulin expression of IEC-6 was detected by using immunostaining and RT-PCR,respectively. as results, TAT-MafA in IEC-6 was dctected after incubation for 12h and insulin was detected in the IEC-6 after incubation for 24th. Thus, TAT-MafA can be transduced into IEC-6 and biological activities could be retained,to induce IEC-6 into insulin positive cells.

关 键 词：蛋白质转导 TAT MAFA 小肠上皮细胞 胰岛素 

分 类 号：Q78[生物学—分子生物学]