TAT透膜肽介导VP3蛋白诱导HepG2细胞凋亡效应的研究

目的探讨TAT透膜肽介导VP3融合蛋白穿透生物膜及诱导HepG2细胞凋亡的效应,为临床应用生物大分子药物治疗肿瘤提供实验基础。方法常规构建TAT-VP3、TAT-GFP及TAT-GFP-VP3的3种融合蛋白的pET28 a原核表达载体,IPTG诱导融合蛋白表达,经镍金属鳌合层析柱纯化获得融合蛋白。通过蛋白转导实验观察其穿透生物膜的效应,利用DAPI染色及TUNEL法观察其诱导HepG2细胞凋亡的效应。结果融合蛋白TAT-GFP及TAT-GFP-VP3可进入体外培养的HepG2细胞,在荧光下可见绿色荧光,融合蛋白TAT-VP3及TAT-GFP-VP3可诱导体外培养的细胞凋亡。结论在VP3的N端加上TAT透膜肽形成的融合蛋白可有效穿透细胞膜,TAT-VP3融合蛋白能诱导HepG2细胞凋亡。

Study on the Effects of Apoptosis of HepG2 Cells Induced by VP3 Inducted by TAT Cell-Penetrating Peptides

Objective The effects of biomembrane penetration of TAT-penetrating peptides and apoptosis of HepG_(2) cells induced by VP3 fusion proteins were investigated to provide experimental basis for the clinical applications of biomacromolecule drug for the biological therapy of tumors.Methods The prokaryote expression vector of pET28a of three kinds of TAT-VP3,TAT-GFP and TAT-GFP-VP3 were constructed by molecular cloning. The fusion proteins were induced by IPTG and purified by nickel affinity gel column chromatography.By protein transduction the effect of biomembrane penetration was observed,and DAPI staining and TUNEL assay were used to detect the apoptotic effects of HepG_(2) cells induced by fusion proteins.Result The TAT-GFP and TAT-GFP-VP3 fusion proteins could penetrate into HepG_(2) cells and display green fluorescences under a fluorescence microscope.The TAT-VP3 and TAT-GFP-VP3 fusion proteins could induce apoptosis in HepG_(2) cells.Conclusion The fusion proteins added by TAT cell-penetrating peptides to N-terminal of VP3 protein could penetrate into cell membrane and the TAT-VP3 fusion protein could induce apoptosis in HepG_(2) cells.