通过RT-PCR从人外周血白细胞中钓取血管生成素(angiogenin,Ang)cDNA,构建诱饵蛋白载体pAS-2-1-Ang,对其自身转录激活活性进行鉴定后,通过酵母双杂交系统筛选人肝细胞cDNA文库,获得两个双阳性克隆.序列分析和同源检索表明所获候选蛋白分别为人上皮细胞素和λ晶体蛋白.构建Ang及候选蛋白标签融合表达载体并共转染COS-7细胞,利用免疫共沉淀和蛋白质印迹方法在哺乳动物细胞中验证了Ang与候选蛋白间的相互作用.为阐明Ang促血管新生的分子机制创造了条件.
The cDNA encoding angiogenin was isolated by RT-PCR from human peripheral white blood cell. The bait protein plasmid of pAS-2-1-Ang was constructed, and it's transcription activity was analyzed. Two positive clones were obtained from human fetal liver cDNA library screened by yeast two-hybrid system. The result of sequence analysis and homology comparison showed the candidate protein was lambda-crystallin and human granulin, respectively. The tag plasmid of angiogenin and candidate protein were contructed and cotransfected into COS-7 cell line. The interaction between angiogenin and candidate protein was identified by the assay of co-immunoprecipitation and Western blotting.
徐东刚,马百坤,李莉,邹民吉,彭善云,王嘉玺.血管生成素相互作用蛋白的发现及其在哺乳动物细胞中的验证[J].生物化学与生物物理进展,2002,29(3):429-433
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