白细胞中与NS5ATP5蛋白结合的蛋白基因的筛选与克隆

Screening and cloning of human gene 5 transactivated by nonstructural protein 5A of hepatitis C virus

目的:采用酵母双杂交体系寻找与NS5ATP5相互作用的白细胞蛋白,以探讨NS5ATP5的生物功能. 方法:应用酵母双杂交系统3,构建NS5ATP5诱饵质粒, 转化酵母AH109,与含人白细胞cDNA文库质粒的酵母Y187进行配合,于涂有x-α-gal营养缺陷型培养基上筛选生长.挑选蓝色克隆,提取此酵母克隆的质粒转化大肠杆菌提取质粒DNA后进行测序,然后进行生物信息学分析. 结果:筛选出10个与NS5ATP5特异性相互作用的克隆,其中1个为金属硫蛋白,1个为热休克蛋白HSP60,1个为主要组织相容性复合体Ⅱ淋巴细胞抗原DQB,5个为人类重排免疫球蛋白λ-轻链,2个是未知功能基因. 结论:初步克隆了NS5ATP5与白细胞结合蛋白基因,对NS5ATP5的功能研究有一定的提示作用;为以后研究这些能与NS5ATP5相互作用的基因在白细胞中的生理功能奠定了基础.

AIM: Human gene 5 transactivated by nonstructural protein 5A of hepatitis C virus (NS5ATP5) is a kind of protein with unknown function from the study with suppression sub-tractive hybridization (SSH). To investigate the biological function of NS5ATP5, we performed yeast-two hybrid to seek for proteins in leucocytes interacting with NS5ATP5. METHODS: NS5ATP5 bait plasmid was constructed by li gating the gene of NS5ATP5 into pGBKT7, then transformed into yeast AH109 (a type), the transformed yeast mated with yeast Y187 (a type) containing leucocyte cDNA library plasmid in 2xYPDA 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 blue colonies, we underwent sequence analysis by bioinformatics. RESULTS: Ten colonies were sequenced. Among them, 8 colonies were genes with known functions and two colonies were new genes. CONCLUSION: The preliminary successful cloning of gene of protein interacting with NS5ATP5 paves the way for studying the physiological function of NS5ATP5 and asso- ciated protein.