6His-VP3融合蛋白的表达、纯化及其捕获细胞中相关蛋白的初步探讨  

Expression and Purification of 6His-VP3 Fusion Protein and Preliminary Research of Its Related Proteins in Tumor Cells

作　　者：王阿慧[1] 孙军[1] 毕昊[1] 彭冬君[1] 田俊[1] 狄勇[1] 宗义强[1] 屈伸[1] 

机构地区：[1]华中科技大学同济医学院基础医学院生物化学与分子生物学系,武汉市430030

出　　处：《医学分子生物学杂志》2007年第2期112-115,共4页Journal of Medical Molecular Biology

基　　金：中华人民共和国教育部博士点基金(No20060487063)~~

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。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.

关 键 词：凋亡素 VP3融合蛋白 相关蛋白 肿瘤特异性 

分 类 号：Q71[生物学—分子生物学]