大眼狮鲈鱼皮肤肿瘤病毒(WDSV)逆转录病毒周期蛋白(OrfA)相互作用蛋白的酵母双杂交筛选  

Screening of Proteins Interacting with the Retroviral-cyclin(OrfA) of Walleye Dermal Sarcoma Virus(WDSV) with Yeast Two Hybrid System

作　　者：徐坤[1] 王亮亮[1] 张婷婷[1] 周罡[1] 张志强[1] 张智英[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《中国生物化学与分子生物学报》2012年第3期267-275,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.30870119)资助项目~~

摘　　要：大眼狮鲈鱼皮肤肿瘤(WDS)在秋季发生春季萎缩的季节性生长特点与大眼狮鲈鱼皮肤肿瘤病毒(WDSV)辅助基因的表达有关.其中辅助基因orfA的表达产物OrfA蛋白可能是1个潜在的肿瘤相关因子,OrfA与细胞周期蛋白cyclin A和cyclin D同源,也被称为逆转录病毒周期蛋白(retroviral-cyclin).为了深入研究OrfA的作用和功能,我们构建了诱饵表达载体pGBKT7-orfA,采用酵母双杂交技术,从HeLa cDNA文库中筛选与其相互的蛋白因子.通过2轮高强度压力的筛选、β-半乳糖苷酶活性验证、回转验证以及测序分析,最终获得了1个与OrfA相互作用的转录因子E2F5.OrfA C端78个氨基酸的多肽或者缺失C端78个氨基酸的OrfA突变体均不能在酵母双杂交系统中与E2F5相互作用.这些发现为进一步研究OrfA在肿瘤萎缩中的功能奠定了基础.The seasonal outbreak of Walleye dermal sarcoma（WDS） starts in the fall and regresses in the spring.This phenomenon is etiologically associated with an accessory gene orfA of WDS virus coding a retroviral-cyclin which is a potential tumor related factor partially homologous to cyclin A and cyclin D.In this research,the full length of orfA was amplified and cloned into a pGBKT7 bait vector,and was used to screen a matchmaker pretransformed HeLa cDNA library,by yeast two hybrid.After two rounds of high stringency selection,β-galactosidase assays and co-transformation of the bait and prey back into AH109,the prey plasmids were rescued and sequenced as the positive interaction partners.We identified an important transcription factor E2F5 that could interact with OrfA.Further mutation studies demonstrated that the C-terminus 78 aa fragment of OrfA alone or its deletion from the full-length abolished the interaction with E2F5 in the yeast two hybrid system.These findings will help the future studies on the functions of OrfA in tumor regression.

关 键 词：大眼狮鲈鱼皮肤肿瘤病毒 逆转录病毒周期蛋白(OrfA) 酵母双杂交 E2F5转录因子 

分 类 号：Q74[生物学—分子生物学] R730.2[医药卫生—肿瘤]