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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 growth characteristics of spring withering in the Great Eyes Lionfish perch skin tumor (WDS) in Autumn are related to the expression of WDSV helper genes, and OrfA protein may be a potential product of orfA , OrfA is homologous to the cyclin A and cyclin D. It is also known as retroviral-cyclin. To further investigate the function and function of OrfA, we constructed a bait expression vector pGBKT7- orfA, and screening the HeLa cDNA library for protein factors interacting with HeLa cDNA library by yeast two-hybrid technique.After two rounds of high-pressure screening, β-galactosidase activity verification, gyrotrmatic verification and sequencing analysis, OrfA interacting transcription factor E2F5.OrfA C-terminal 78 amino acid polypeptide or lacking C-terminal 78 amino acid OrfA mutant can not interact with E2F5 in yeast two-hybrid system.These findings are for further study OrfA in tumor shrinkage The function has laid the foundation.