酵母双杂交技术筛选p7TP2蛋白结合肝细胞蛋白的编码基因  

Screening of proteins binding to p7TP2 protein from liver cDNA library by yeast two-hybrid technique

作　　者：袁菊[1] 成军[1] 洪源[1] 靳亚平[2] 韩莉[1] 陶明亮[1] 李越[1] 王琦[1] 

机构地区：[1]北京地坛医院传染病研究所,100011 [2]西北农林科技大学动物科技学院

出　　处：《中华实验和临床感染病杂志（电子版）》2007年第2期72-75,共4页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

基　　金：国家重点基础研究项目(2004CB518908)

摘　　要：目的筛选HCVp7蛋白反式调节蛋白p7TP2的结合蛋白,探讨HCV的致病机制及新蛋白p7TP2的生物学功能。方法应用酵母双杂交系统3,将多聚酶链反应(PCR)法扩增的p7TP2基因连接入酵母表达载体pGBKT7中构建诱饵质粒,转化单倍体酵母细胞AH109并在其内表达,然后与转化了人肝细胞文库质粒pACT2的单倍体酵母细胞Y187进行配合,在营养缺陷型培养基和X-α-半乳糖(X-α-gal)上进行双重筛选,提取阳性酵母菌落的质粒转化大肠埃希菌氨苄青霉素-LB平板上选择并测序,结果在GenBank中进行生物信息学分析。然后将阳性文库质粒与诱饵质粒回交,以证实其相互作用。结果筛选出与p7TP2结合的蛋白基因10个,其中包括金属硫蛋白2A、载脂蛋白H、蛋白C、果糖二磷酸醛缩酶B、配对免疫球蛋白样2型受体α、CTL1基因类胆碱运输蛋白等已知功能蛋白基因相互作用,回交试验已经证实其相互作用。结论由本研究结果发现p7TP2蛋白可以结合一系列不同功能的肝细胞蛋白,作为一种新基因,对它的进一步作用机制的研究将为阐明HCV感染及慢性肝炎、肝纤维化和肝癌的病理机制提出新的思路。Objective To study the pathogenesis of HCV and the biological functions of a novel HCV p7 trans-regulated protein（p7TP2）. Methods p7TP2 bait plasmid was constructed by ligating the p7TP2 cDNA with yeast expression plasmid pGBKT7, then pGBKT7-P7TP2 was transformed into yeast cells AH109 （a type）. Thereafter, the transformed yeast cells were amplified and mated with yeast cells Y187 （αtype） containing liver cDNA library plasmid pCAT2 in 2×YPDA medium, diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） containing X-α-gal for selecting two times. Plasmids of true positive blue colonies were extracted and analyzed by DNA sequencing and blast search in GenBank database. Results Eleven genes, including MT2A, APOH, protein C, eukaryotic translation elongation factor 1α 1, paired immunoglobin-like type 2 receptor α, CTL1 gene for choline transporter-like protein, etc, have been found binding with p7TP2 protein. Conclusions These results show that p7TP2 protein could bind to proteins of inhibiting blood clotting directly or indirectly. All these brought some new clues for studying the biological functions of novel gene p7TP2 and the pathogenesis of HCV.

关 键 词：HCVp7 酵母双杂交 回交试验 抗凝血 

分 类 号：R346[医药卫生—基础医学]