酵母双杂交技术筛选与HCMV-UL146编码产物相互作用蛋白

Identification of proteins interacting with HCMV UL146 encoded products by yeast two-hybrid assay

目的应用酵母双杂交技术,在人胎脑文库中筛选与人类巨细胞病毒(human cytomegalovirus,HCMV)UL146基因编码蛋白相互作用蛋白,以探讨HCMV-UL146的生物功能。方法应用酵母双杂交系统,构建pGBKT7-UL146诱饵质粒,转化酵母AH109,与含人胎脑cDNA文库质粒的酵母相互作用,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进行测序分析。结果 pGBKT7-UL146构建成功。经严格筛选,共有200余个阳性克隆,分离测序80个克隆,成功筛选出30个与HCMV-UL146特异性相互作用的蛋白。结论 HCMV-UL146与多种蛋白有相互作用,提示其生物学功能复杂。此结果为进一步研究HCMV-UL146基因功能提供依据。

【Objectives】 To search for proteins interacting with human cytomegalovirus（HCMV） UL146 encoded protein with the yeast two-hybrid system.【Methods】 A human fetus brain cDNA library was screened with the pGBKT7-UL146 recombinant plasmid as a bait to find proteins interacting with the UL146 protein.The plasmids in positive yeast clones were selectively identified by PCR analysis and DNA sequencing.【Results】 The pGBKT7-UL146 bait plasmid was constructed successfully and co-transformed with a human fetus brain cDNA library into yeast cells.There were more than two hundreds positive yeast clones on-Ade/-His/-Leu/-Trp high-stringency selection plates.The pACT2-cDNA plasmids in 80 yeast clones were isolated and sequenced.At least 30 proteins interacting with the HCMV UL146 were identified.【Conclusion】 The HCMV-UL146 encoded protein can interact with several distinct proteins.The results suggest that HCMV-UL146 has a wide variety of biological functions.