应用白细胞表达型cDNA文库的酵母双杂交技术筛选丙型肝炎病毒非结构蛋白5A的结合蛋白

Screening and Identification of Hepatitis C Virus Nonstructural Protein 5A-interacting Proteins from Leukocyte by Yeast Two-hybrid Technique

目的:研究丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)与白细胞来源蛋白的相互作用,探讨HCVNS5A对于病毒致病的机制及对机体免疫的影响。方法:构建HCV NS5A的酵母细胞表达载体,采用酵母双杂交系统筛选人白细胞cDNA文库,利用核苷酸数据库及生物信息学技术,对于筛选结果进行分析。结果:获得了53个与NS5A特异性结合的阳性克隆,包括junB原癌基因、Mob-1、制瘤素M、肌球蛋白IF、小囊泡结合膜蛋白的结合蛋白等16种已知蛋白基因,和2个未知功能基因。结论:NS5A可与一些白细胞特异性蛋白和信号转导组分发生结合,为HCV所引起的肝内外疾病机制的研究提供线索。

Objective: To study the relationship of hepatitis C virus (HCV) non-structure protein 5A and proteins from leukocytes and investigate the pathogenesis of HCV.Methods: HCV NS5A bait plasmid was constructed into pGBKT7, then transformed into yeast AH109 (a type), the transformed yeast mated with yeast Y187 (α type) containing leukocyte cDNA library plasmid in 2×YPDA medium.Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from positive colonies, we underwent sequence analysis by bioinformatics.Results: Fifty-two clones were obtained and sequenced, these proteins included jun B pro-to-oncogene, oncostatin M, RAN binding protein 9, mob-1, etc.Conclusion: NS5A interact with proteins specific to leukocyte and components of signal transduction, that paved the way for further studying of pathogenesis of HCV NS5A.