穿膜肽TAT-p38αG融合基因的构建、表达及穿膜特性的鉴定  

Construction,expression of TAT-p38αG and its capability of transduction into endothelial cells in vitro

作　　者：张家平[1] 应希[2] 陈渝[1] 张琼[1] 黄跃生[1] 

机构地区：[1]第三军医大学西南医院:全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆400038 [2]第三军医大学西南医院全军眼科中心,重庆400038

出　　处：《第三军医大学学报》2008年第1期15-18,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30300366)~~

摘　　要：目的构建穿膜肽TAT-p38αG融合基因,进行原核表达及穿膜特性鉴定。方法提取人EVC304细胞总RNA,巢式PCR扩增p38αG基因片段,T-A克隆,测序证实片段无误后,将p38αG基因片段亚克隆入原核表达载体pTAT-HA,获得pTAT-p38αG融合基因表达载体。将表达载体转化大肠杆菌,IPTG诱导表达获得TAT-p38αG融合蛋白,并行Ni-NTA柱过柱和超滤纯化,抗His抗体免疫印迹法鉴定。采用荧光素FITC体外标记TAT-p38αG融合蛋白,与EVC304细胞共培养,荧光显微镜下观察其穿膜特性。结果成功构建了融合基因原核表达载体pTAT-p38αG,并表达出大小约30×103的蛋白产物,与融合蛋白TAT-p38αG理论计算值相符。细胞实验结果显示,TAT-p38αG能呈浓度依赖性方式转导进入EVC304细胞。结论TAT-p38αG融合蛋白具有良好的穿膜特性,研究为下一步鉴定TAT-p38αG融合蛋白对p38α激酶的抑制作用奠定了良好的实验基础。Objective To construct a recombinant vector containing TAT-p38αG fusion gene HIV transcription factor TAT and a docking motif of p38α MAP kinase and investigate the transduction capability of the expressed TAT-p38αG fusion protein into endothelial cells. Methods The p38αG fragment was amplified by PCR, cloned into pMD18-T vector, and then subcloned into pTAT-HA vector. The constructed vector pTAT- p38αG was further transfected into E. coli DH5α, and then into BL21 after sequencing. The expression of TAT-p38αG protein was induced by IPTG. After purification by Ni-NTA column and uhrafiltration, the fusion protein was labeled by FITC, and cocultured with endothelial cell line EVC304. The transduction capability of TAT-p38αG into EVC304 cell was then determined. Results The recombinant vector containing TAT-p38αG was constructed and TAT-p38αG protein was expressed successfully. FITC labeled TAT-p38αG protein was able to be transducted into EVC304 cells in a dose-dependent manner. Conclusion TAT-p38αG protein shows a high capability of transduction into endothelial cells, which enable us to further investigate its possible inhibitory role in p38α kinase activity.

关 键 词：p38激酶α亚型 穿膜肽 融合基因 

分 类 号：R345[医药卫生—基础医学] R394-33