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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(Tat11)的2条寡核苷酸单链,两端分别引入BamHⅠ和HindⅢ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-Tat11-TK的BL21菌通过IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,免疫组化分析重组蛋白Tat11-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、Tat11-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Tat11-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Ni2+螯合柱亲和纯化获得的Tat11-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Tat11-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,150μmol/L)能有效抑制HepG2细胞生长。结论:Tat11-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 investigate the killing effect of HIV-1 trans-activating protein Tat fused with HSV1-TK on hepatoma cells. METHODS: Two single-stranded oligonucleotides encoding HIV-Tat47-57 (Tat11) were synthesized. Two ends of BamHⅠand HindⅢ were introduced into the two ends of the single-stranded oligonucleotide. The two oligonucleotides were annealed to form an oligonucleotide duplex. The 15% Acrylamide gel electrophoresis to determine the annealing effect; HSV1-TK gene was amplified by PCR using r-pAs16Dr as a template and cloned into prokaryotic expression vector pET-32. The BL21 strain containing pET-32c-Tat11-TK was induced to express by IPTG. The expressed product was purified by affinity chromatography on Ni2 + chelate column. The membrane-bound properties of the recombinant protein Tat11-TK were analyzed by immunohistochemistry. Through the ability of membrane penetration, the killing effect of TK / GCV and Tat11-TK / GCV on HepG2 cell line was detected and analyzed. Results: The SDS-PAGE of the expressed product showed a band with a molecular weight of 60700, which was in line with the theoretical value of Tat11-TK and the expression of the tagged fusion protein and was proved to be expressed as an inclusion body. Tat11 obtained by affinity purification with Ni2 + chelating column -TK recombinant fusion protein was confirmed by immunohistochemistry to bind to the surface of hepatocellular carcinoma cells. The results of western blotting showed that Tat11-TK could efficiently penetrate the cell membrane into HepG2 cells, and at the same time, low concentration prodrug ganciclovir (GCV, 150μmol / L) can effectively inhibit HepG2 cell growth. CONCLUSION: Tat11-TK is highly expressed in prokaryotic system with strong ability of transmembrane penetration and prodrug enzyme activity.