酵母双杂交技术筛选人白细胞中与乙型肝炎病毒X蛋白结合蛋白基因

Screening of lymphocyte proteins interacting with hepatitis B virus X antigen by yeast-two hybrid technique

目的:筛选并克隆人白细胞中与乙型肝炎病毒(HBV)X 蛋白(HBxAg)相互作用蛋白的基因,进一步探讨 HBxAg 的生物学功能.方法:用多聚酶链反应(PCR)法扩增 HBxAg 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,然后与转化了人淋巴 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)上双重筛选阳性菌落并测序,进行生物信息学分析.结果:成功克隆出 HBxAg 基因并在酵母细胞中表达,配合后选出在四缺(SD/-Trp-Leu-Ade-His)培养基和在铺有 X-α-gal 的四缺培养基上均能生长,并变成蓝色的真阳性菌落50个,其中含真核翻译延伸因子2基因20个,真核翻译延伸因子1基因1个,真核翻译起始因子3基因2个,急性髓样白血病 MTG8蛋白基因2个,髓样关联分化蛋白基因1个,髓样分化主反应蛋白基因1个,皮肤 T 细胞淋巴瘤肿瘤抗原基因1个,大组织相容性复合物 Ib 抗原基因3个,白细胞抗原 CD37基因1个,慢性白细胞性白血病相关抗原基因1个,α白细胞整联蛋白基因1个,生长停止和 DNA 损伤诱导因子34基因1个,转录变异因子1基因2个,蛋白磷酸酶1调控抑制因子15A 基因1个,单核细胞系胞苷脱氨酶基因1个,巨噬细胞钙离子依赖型凝集素2基因1个,KIAA1949蛋白基因3个,尿激酶型血浆素原激活剂基因1个,GDP 分解抑制因子基因3个,软骨素4磺基转移酶基因1个,锌指蛋白1A 亚目基因1个和未知蛋白基因1个.结论:成功克隆出乙型肝炎病毒 X 蛋白的结合蛋白,为进一步研究 HBxAg 的生物学作用提供了新的线索.

AIM:To investigate the biological functions of hepatitis B virus X protein(HBxAg). METHODS:The HBxAg gene was amplified by polymerase chain reaction(PCR)and HBxAg bait plasmid was con- structed by using yeast-two hybrid system 3,then trans- formed into yeast AH109.The transformed yeast mated with yeast Y187 containing lymphocytes cDNA library plasmid in 2×YPDA medium.Diploid yeast was plated on synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)and syn- thetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)con- taining x-α-gal for selecting two times and screening.Af- ter extracting and sequencing of plasmid from blue colonies, we underwent analysis by bioinformatics. RESULTS:A total of 50 colonies were sequenced.Among them,20 colonies were eukaryotic translation elongation factor 2,1 eukaryotic translation elongation factor 1,2 eukaryotic translation initiation factor 3,2 protein MTGR1a,1 myeloid associated differentiation protein,1 myeloid differentiation primary response protein MYD116,1 CTCL tumor antigen HD-CL-08,3 MHC class Ib antigen(HLA-E),1 leukocyte antigen CD37,1 aCLL-associated antigen KW-6,1 lym- phocyte function-associated antigen 1,1 growth arrest and DNA-damage-inducible 34,2 transcript variant 1,1 protein phosphatase 1,regulatory inhibitor subunit 15 A,1 cyti- dine deaminase,1 macrophage lectin 2(calcium dependent)(HML2),3 KIAA1949 protein,1 urokinase-type plasminogen activator receptor,3 GDP dissociation inhibitor, 1 chondroitin 4-sulfotransferase,1 zinc finger protein,sub- family 1A and a new gene with unknown function. CONCLUSION:Genes of HBxAg-interacting proteins in lym- phooltes are successfully cloned and the results bring some new clues for studying the biological functions of HBxAg and associated proteins.