酵母双杂交技术筛选α干扰素结合蛋白的基因  

Screening of interferonαbinding proteins by yeast-two hybrid technique

作　　者：曲建慧[1] 成军[1] 张玲霞[1] 钟彦伟[1] 杨媛[1] 郭江[1] 张黎颖[1] 刘妍[1] 王琳[1] 戴久增[1] 徐东平[1] 

机构地区：[1]解放军第302医院病毒性肝炎研究所,北京100039

出　　处：《中华传染病杂志》2006年第4期235-238,共4页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金(30371288);第35批博士后科学基金(2004035045)

摘　　要：目的筛选人肝细胞cDNA文库中与α干扰素(IFNα)蛋白具有相互作用的蛋白基因。方法用聚合酶链反应(PCR)扩增IFNα基因,连接入酵母表达载体pGBKT7中构建诱饵质粒,转染酵母细胞AH109,Western blot证明IFNα蛋白能够在AH109中表达。然后将AH109与转染了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和X-α-半乳糖(X-α-gal)上进行双重筛选阳性菌落,提取质粒后转化DH5α大肠埃希菌并经氨苄西林抗性筛选,提取单克隆菌落质粒,酶切鉴定正确者进行测序,再行生物信息学分析。结果成功克隆化IFNα基因并在酵母细胞中表达,应用酵母双杂交筛选出阳性菌落34个,经生物信息学分析,排除读码框架不正确者,最后得到8种已知基因:玻璃体连接蛋白、纤维蛋白原α多肽、人类免疫缺陷病毒(HIV)1Tat相互作用蛋白2、精氨酸酶、NADH脱氢酶1β亚复合物、转铁蛋白受体2α、酒精脱氢酶IBβ多肽、肝细胞癌蛋白(HCC-1);2种染色体基因:人染色体17,克隆RP11-350B3,人染色体10,克隆RP11-351O1。结论成功克隆出IFNα基因,并从肝细胞cDNA文库中筛选出8种能与IFNα具有结合作用的蛋白基因。Objective To screen proteins binding with interferon α（IFNα）from human hepatic cDNA libraty by yeast two hybrid technique. Methods The IFNα gene was amplified by polymerase chain reaction （PCR） and constructed into pGBKT7 vector as the bait plasmid in yeast two hybrid system3, pGBKT7-IFNα was then transfected into yeast AH109. The transfected yeast were mated with yeast Y187 containing liver cDNA library plasmid in 2 × YPDA medium. Diploid yeast was 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. After plasmid extracting and enzyme cutting analysis, the blue colonies were performed sequence analysis, the results were analyzed by bioinformatics. Results IFNα gene was successfully cloned and expressed in yeast cells. Thirtyfour positive colonies were obtained using yeast-two hybrid technique. After sequence analysis,eight clones were found may have a binding effect with IFN protein. Conclusions IFNα genes was successful cloned and eight proteins that could bind with IFNα protein were also screened.

关 键 词：干扰素Α 酵母菌 双杂交系统技术 克隆 分子 基因 

分 类 号：R346[医药卫生—基础医学]