乙型肝炎病毒截短型表面抗原中蛋白结合蛋白的筛选  

Screening and identification of proteins interacting with C-terminally truncated middle surface protein of hepatitis B virus

作　　者：田江克[1] 成军[2] 刘妍[3] 崔玉芳[1] 纪冬[3] 王琳[3] 程勇前[3] 钟彦伟[3] 徐东平[3] 

机构地区：[1]解放军军事医学科学院放射与辐射医学研究所免疫学研究室,北京市100850 [2]北京地坛医院传染病研究所,北京市100011 [3]解放军第302医院传染病研究所病毒性肝炎研究中心,北京市100039

出　　处：《世界华人消化杂志》2006年第24期2382-2385,共4页World Chinese Journal of Digestology

基　　金：北京市自然科学基金资助项目;No.5042024~~

摘　　要：目的:用酵母双杂交技术筛选白细胞与乙型肝炎病毒截短型表面抗原中蛋白(MHBst)结合蛋白．方法:PCR扩增MHBst基因,连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达．后与转化了人白细胞文库质粒的酵母细胞Y187进行配合,在营养缺陷型培养基(SD／-Trp-Leu-His-Ade)和X-α-半乳糖(X-α-gal)上进行双重筛选阳性菌落增菌后提出质粒转化入大肠杆菌(DH5α),并经氨苄青霉素抗性筛选,提取单克隆菌落质粒,酶切鉴定,序列测定后进行生物信息学分析．结果:应用酵母双杂交技术筛选出阳性克隆8个,经生物信息学分析,排除读码框架不正确者,最后得到7种已知基因,分别为ADP核糖基因子2种,RAB6相互作用蛋白(RAB6IP1)1种,CCR4-NOT转录复合体亚基10(CCR4- NOT-10)1种,脂多糖蛋白结合蛋白(LBP)1种,醛缩酶B(ALDOB)1种,补体成分3(C3)1种,丙酮酸脱氢酶(PDH)1种．结论:成功克隆出MHBst结合蛋白．AIM： To screen and identify proteins interacting with C-terminally truncated middle surface protein of hepatitis B virus （MHBst） from human leukocyte cDNA library by yeast two-hybrid system. METHODS： The MHBst fragment was amplified by polymerase chain reaction （PCR） and MHBst binding protein bait plasmid pGBKT7-MHBst was constructed by yeast-two hybrid system,Then plasmid pGBKT7-MHBst was transformed into yeast AH109 cells and the expression of MHBst was detected by SDS-PAGE and Western blotting assay. The transformed yeast cells were mated with yeast Y187 containing leukocyte cDNA library plasmid. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） and quadruple dropout （QDO） medium containing X-α-gal for selection. Subsequently, the plasmids of the blue colonies were extracted, transformed into E coli （DH5α）, and then selected by ampicillin. After extraction and digestion, the plasmids were analyzed by DNA sequencing and bioinformafics. RESULTS： MHBst proteins were successfully cloned and expressed in yeast cells. Eight genes in positive colonies were obtained by yeast-two hybrid technique, including two ADP-ribosylation factor 1, one RAB6 interacting protein 1, one CCR4-NOT transcription complex subunit 10, one lipopolysaccharide binding protein, one aldolase B, one complement component 3 and one pyruvate dehydrogenase. CONCLUSION： MHBst binding proteins in leukocytes are successfully cloned.

关 键 词：酵母双杂交技术 MHBst 结合蛋白 克隆 

分 类 号：R392[医药卫生—免疫学]