从人白细胞cDNA文库筛选凋亡素相互作用蛋白

Screening of Protein Interacting with Apoptin by Yeast Two-hybrid from Human Leucocyte cDNA Library

来源于鸡贫血病毒的小分子蛋白 凋亡素 (apoptin)能够选择性诱导肿瘤细胞凋亡 ,为研究其选择性诱导肿瘤细胞凋亡的分子机制 ,利用酵母双杂交系统筛选从人白细胞cDNA文库筛选apoptin相互作用蛋白 ,核酸序列分析及同源性检索表明 ,其中一个与ABP2 80 (actin bindingprotein 2 80 )有高度同源性 .细胞免疫共沉淀实验结果显示 :在哺乳动物细胞水平仍能够检测到apoptin与ABP2 80片段的特异的相互作用 .分别构建缺失C端 11个氨基酸、中间 33～ 46位氨基酸和二者均缺失的apoptin的 3个突变体 ,突变体与ABP2 80相互作用研究表明 :apoptin的 33～ 46位氨基酸 (核外运信号 )对于apoptin与ABP2 80的相互作用是必需的 ,而C端核定位信号 /DNA结合序列对于apoptin与ABP2

Using yeast two-hybrid system to screen the protein interacting with apoptin from human leucocyte cDNA library, four clones interacting with apoptin were identified. One of them was homologue with ABP280 (actin-binding protein), ABP280 is a dimeric actin crossing protein and plays a key role in stabilizing the membrane-cytoskeleton. Cell co-immunoprecipitation showed that apoptin could bind to ABP280 in mammalian cells. Apoptin mutants T1, T2 and T3 lack the C-terminal 11 amino acid, 33 similar to 46 amino acid and both respectively. Apoptin mutants T2 and T3 failed to interact with ABP280, which revealed that its 33 similar to 46 amino acid was pivotal for the interaction. Apoptin mutant T1 still interacted with ABP280, which revealed that its C-terminal 11 amino acid was not essential for the interaction.