摘要
目的:寻找可能与锌指转录因子ZNF580存在相互作用的蛋白。方法:以ZNF580基因开放阅读框作为模版,PCR扩增后连接入酵母表达质粒pGB。诱饵质粒pGB-ZNF580经测序验证后转化酵母菌株Y190,人胎脑cDNA文库亦转化到能稳定表达诱饵蛋白的Y190酵母菌株中,并铺到含有营养缺陷型培养基(SD/-Trp/-His/-Leu)的培养皿上进行初步筛选。所得阳性克隆再进行β-半乳糖苷酶克隆转移滤纸实验进一步去除假阳性。随机挑取部分阳性克隆,逐一转化入含有诱饵质粒的Y190酵母菌进行一对一验证。分离阳性克隆质粒测序,并应用生物信息学方法分析阳性克隆cDNA编码的蛋白。结果:确定了14种与ZNF580可能存在相互作用的蛋白。结论:初步探讨了ZNF580的功能及其可能参与的信号转导通路,为进一步研究转录因子ZNF580参与动脉粥样硬化的机制奠定了实验基础。
AIM： To identify the possible proteins interacting with zinc finger transcription factor ZNF580. METHODS： The open reading frame of ZNF580 gene was amplified by PCR and ligated into plasmid pGB. The bait plasmid pGB-ZNF580 was transformed into Y190 yeast strain after sequencing. Human fetal brain cDNA library was also transformed into the bait yeast Y190 that could express the bait protein. The co-transformants were plated onto SD/-Trp/-Leu/-His selective medium. Recognition of positive colonies was performed by β-galactosidase colony-lift filter assay. Some positive plasmids were transformed into the bait yeast separately further to eliminate the false positive ones. The inserted cDNAs were extracted and analyzed for homology using the BLAST program against the GenBank/EMBL/DDBJ non-redundant sequence database. RESULTS： Fourteen proteins were identified to possibly interact with ZNF580. CONCLUSION： The possible signal pathway in which ZNF580 may be involved was initially explored.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2012年第6期1120-1123,共4页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81170106)
天津市自然科学基金资助项目(No.10JCYBJC26500)
武警后勤学院资助项目(No.WYM2010-5)
