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

张家平; 应希; 陈渝; 张琼; 黄跃生

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

更新时间：2026-01-09

- Construction, expression of TAT-p38αG and its capability of transduction into endothelial cells in vitro
- Journal of Army Medical University Vol. 30, Issue 1, Pages: 15-18(2008)
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  CLC： R345;R394-33;R394.2
- Published：2008
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张家平,应希,陈渝,张琼,黄跃生.穿膜肽TAT-p38αG融合基因的构建、表达及穿膜特性的鉴定[J].陆军军医大学学报(原第三军医大学学报),2008,30(01):15-18.

　ZHANG Jia-ping,YING Xi,CHEN Yu,ZHANG Qiong,HUANG Yue-sheng.Construction, expression of TAT-p38αG and its capability of transduction into endothelial cells in vitro[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(01):15-18.
张家平,应希,陈渝,张琼,黄跃生.穿膜肽TAT-p38αG融合基因的构建、表达及穿膜特性的鉴定[J].陆军军医大学学报(原第三军医大学学报),2008,30(01):15-18. DOI：

　ZHANG Jia-ping,YING Xi,CHEN Yu,ZHANG Qiong,HUANG Yue-sheng.Construction, expression of TAT-p38αG and its capability of transduction into endothelial cells in vitro[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(01):15-18. DOI：

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30×10

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。

Abstract

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 TATp38αG 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 ultrafiltration

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.

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