Tat蛋白转导结构域介导HSV1-TK导入肝癌细胞及其效应  

Transfer domain of HIV-1 Tat mediates HSV-1 thymidine kinase into hepatic cancer cells: the mediating effect and efficacy

作　　者：曹利民[1] 王炜煜[2] 赵晓蓉[1] 叶庆[1] 雷萍[1] 刘静[1] 代维[1] 朱荫昌[3] 司进[3] 沈关心[1] 

机构地区：[1]华中科技大学同济医学院免疫学系,武汉430030 [2]华中科技大学同济医学院同济医院普外科,武汉430030 [3]江苏省寄生虫病防治研究所,无锡214064

出　　处：《中国肿瘤生物治疗杂志》2006年第3期162-166,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家重点基础研究发展规划("973"计划)项目(2002CB513109);卫生部科研基金(WKJ2004-2-013)

摘　　要：目的：探讨HIV-1编码的反式激活蛋白Tat与HSV1-TK融合表达对肝癌细胞的杀伤效果。方法：合成编码HIV-Tat47-57（Tat 11）的2条寡核苷酸单链，两端分别引入BamH Ⅰ和Hind Ⅲ两个酶切位点，退火形成寡核苷酸双链，15％非变性聚丙烯酰胺凝胶电泳判断退火效果；以r-pAs16Dr为模板，通过PCR扩增HSV1-TK基因，定向克隆至原核表达载体pET-32中。将含pET-32c-Tat 11-TK的BL21菌通过IPTG诱导表达，表达产物用Ni^（2＋）螯合柱亲和纯化，免疫组化分析重组蛋白Tat 11-TK的膜结合特性，蛋白印迹技术分析该融合蛋白的穿膜能力，检测和分析TK／GCV、Tat 11-TK／GCV对肝癌细胞株HepG2的杀伤作用。结果：表达产物SDS-PAGE在相对分子质量60700左右显示条带，符合Tat 11-TK与表达标签融合蛋白的理论值，并证明以包涵体的形式表达；通过Ni^（2＋）螯合柱亲和纯化获得的Tat 11-TK重组融合蛋白，经免疫组化证实能结合到肝癌细胞表面，蛋白印迹结果说明Tat 11-TK能有效穿过细胞膜进入HepG2细胞内，同时应用低浓度前药更昔洛韦（GCV，150 μmol／L）能有效抑制HepG2细胞生长。结论：Tat 11-TK在原核系统获得高效表达，具有较强的穿膜能力和前药酶活性。Objective： To explore the killing effect of the fusion protein （ HSV1-TK fused with an 11-amino-acid peptide of the basic domain of the HIV-1 Tat protein, Tat11 ） on hepatoma cell line HepG2. Methods： Two single oligonucleotide chains encoding HIV-Tat47-57 （Tat11 ）were synthesized and were used for producing double strand oligonucleotide through introducing BamH Ⅰ and Hind Ⅲ and annealing, 15% non-denaturing polyacrylamide gel electrophoresis was used for analysis of the annealing result. The full length cDNA encoding HSV1-TK was amplified from r-pAs16Dr by PCR. Then the 2 fragments were sub-cloned into a preukaryotic expression vector （pET-32c）. A single colony of E. coli BL21 containing the plasmid pET-32c-Tat11-TK was innoculated LB broth, diluted 1/100 into 1 000 ml LB broth, and was then treated with 1 mmol/L IPTG. The recombinant Tat11-TK was purified with Ni^（2＋） chelating HiTrap HP column and its intercellular translocation ability was evaluated by immnunohistochemistry and Western blot. Results： The recombinant Tat11-TK protein showed bands at about 60 700, which was in accordance with the theoretical value of the fusion protein, also proved the presence of inclusion body. The result of immnunohistochemistry showed that Tat11-TK could bind to the cell surface and Western blot showed that it could also effectively enter into the HepG2. It was also found that HepG2 was more sensitive to GCV（ 150 μmol/L） in the presence of Tat11-TK. Conclusion： The fused protein Tat11-TK can be highly expressed in a preukaryotic system and has potent ability for membrance perforation and enzyme trafficking.

关 键 词：HIV-1 TAT HSV1-TK 蛋白转导 肝癌 基因治疗 

分 类 号：R730[医药卫生—肿瘤]