【摘要】 目的研究人肝癌细胞HepG2与人胎肝细胞L-02中与VP3蛋白相互作用的蛋白组分,以探讨VP3特异性诱导肿瘤细胞凋亡的机制。方法采用PCR方法克隆vp3的DNA片段,将其插入质粒pET-28a(+)构建含6个组氨酸标签6His-VP3融合蛋白的原核表达载体。利用亲和层析技术制备纯化的6His-VP3融合蛋白作为诱饵,分别从肿瘤细胞HepG2及正常细胞L-02的总蛋白中,通过pull-down技术捕获与VP3相互作用的特异性蛋白质组分。结果获得了高纯度的6His-VP3融合蛋白;以其作为诱饵从HepG2细胞和L-02细胞中捕获到的与VP3作用的相关蛋白质组分存在差异。结论肿瘤细胞HepG2及正常细胞L-02中,与VP3作用蛋白的差异与其在不同细胞中的生物学行为相对应。更多还原

【Abstract】 Objective To examine the proteins interacting with 6His-VP3 in HepG2 and L-02 cells in order to uncover the mechanism of VP3-induced tumor specific apoptosis.Methods Vp3 fragment amplified by PCR was inserted into the plasmid pET-28a(+) to construct the prokaryotic expression vector pET-28a(+)-vp3. Using chromatographically purified 6His-VP3 fusion protein as the bait, the VP3-related proteins from cells were captured by employing pull-down technique. Results Polyacrylamide gel electrophoresis showed that the highly pure 6His-VP3 was obtained. The VP3-related proteins captured by using the pure protein as bait from the tumor cell line HepG2 and from normal cell line L-02 were different. Conclusions The differences in the actions with VP3-related proteins in tumor cells and normal cells correspond to the biological actions of VP3 in different cells.更多还原