利用酵母双杂交系统筛选与人巨细胞病毒皮层蛋白pUL23相互作用的病毒编码蛋白

人巨细胞病毒(HCMV) UL23基因编码病毒皮层蛋白,该基因缺失时,病毒在人包皮成纤维细胞(HFF)中的繁殖速度加快.为进一步阐述HCMV UL23基因编码产物 pUL23的功能及调控机制,采用鸟枪法构建了融合于GAL4活性区域的HCMV Towne株 基因组随机表达文库.利用酵母双杂交技术,以pGBKT7 -UL23为诱饵质粒,从构建 的HCMV基因组表达文库中筛选到与pUL23相互作用的病毒编码蛋白pUL24. GST-pull down实验和免疫共沉淀实验进一步确认两种病毒蛋白之间的相互作用.结果 表明,构建的HCMV基因组表达文库能够用于GAL4酵母双杂交系统筛选与诱饵蛋白相互作用的病毒自身编码蛋白.病毒蛋白pUL23和pUL24之间具有相互作用,这为进一 步阐述pUL23在HCMV感染过程中的功能提供依据.该研究为揭示HCMV病毒感染机制奠定了基础.

Screening of Human Cytomegalovirus Tegument Protein pUL23 Interacting Partner via Yeast Two-hybrid System

Human cytomegalovirus(HCMV) UL23 gene codes a tegument protein pUL23, and the UL23 deletion HCMV mutant exhibited enhanced growth in HFF cells. In order to provide an effective tool for further study of the function and regulatory mechanism of HCMV pUL23, a random genomic library of HCMV strains Towne fused to the GAL4 activation domain was constructed by shot-gun method. Using this library, we performed yeast two-hybrid screening with a bait plasmid encoding a fusion of the GAL4 DNA binding domain (GAL4-BD) with the UL23 open reading frame. One of the interacting clones corresponds to HCMV protein pUL24. GST-pull down and co-immunoprecipitation assays confirmed the interaction between these two vial proteins. These results demonstrated that the viral genomic library could be applied to GAL4 yeast two-hybrid assay for screening HCMV proteins which interact with the bait protein. And the interaction between HCMV proteins pUL23 and pUL24 will be further characterized to elucidate the functions of pUL23 during HCMV infection. It was laid a foundation for further study of the infection mechanism of HCMV.