人巨细胞病毒UL141编码蛋白相互作用蛋白的筛选和分析

Screening and Analysis of the Proteins Interacting with Human Cytomegalovirus UL141 Encoded Protein

目的应用酵母双杂交技术筛选人胎脑cDNA文库中与人巨细胞病毒(HCMV)UL141编码蛋白相互作用的蛋白,以进一步研究HCMV UL141的生物学功能,为揭示HCMV先天感染机制奠定基础。方法采用PCR技术扩增HCMV-UL141基因片段,将其插入到pGBKT7载体中,构建成诱饵质粒pGBKT7-UL141。然后将pGBKT7 UL141转化到酵母感受态细胞AH109中,于一缺培养基(SD/Trp)上筛选生长。再将人胎脑cDNA文库质粒转化到含有pGBKT7-UL141质粒的AH109菌株中,在四缺培养基(SD/Trp/Leu/His/Ade)中筛选相互作用的人胎脑cDNA文库蛋白。通过显色反应挑选显蓝色的阳性克隆,提取相应质粒将其转化到大肠杆菌中,并进行回转验证。最后对筛选得到的阳性克隆进行测序及生物信息学分析。结果诱饵质粒pGBKT7UL141构建成功。筛选获得6个克隆与HCMV-UL141编码蛋白相互作用,其中1个基因编码人类染色质解旋酶DNA结合蛋白3(CHD3)。结论成功应用酵母双杂交系统筛选出与HCMV-UL141编码蛋白相互作用的人体组织蛋白——CHD3,提示UL141蛋白可能与病毒的免疫逃避,干扰正常细胞的生长、分化相关。

Objective To screen the proteins interacting with human cytomegalovirus （HCMV） UL141 encoded protein in human fetus brain cDNA Library,so as to improve the further biological function study of HCMV UL141,and provide a foundation for revealing the pathogenic mechanism of HCMV infection.Methods The DNA fragment of HCMV UL141 was amplified by polymerase chain reaction,and cloned into yeast-expressing vector pGBKT7to construct the vector pGBKT7-UL141.Then the recombinant plasmid pGBKT7-UL141 was transformed into the yeast AH109 competent cell.The transformed yeast cells were seeded on nutrient deficiency medium-Trp,and then human fetus brain cDNA library plasmid was also transformed into the yeast cell AH109 containing the pGBKT7-UL141 plasmid.The yeast cells were plated on nutrient deficiency medium-Ade/-His/-Leu/-Trp.Blue positive clones were selected by chromogenic reactio7,and the corresponding yeast plasmids was extracted and transformed into E.coli for verification.Finally,positive clones were sequenced and analyzed by bioinformatic methods.Results The bait plasmid pGBKT7-UL141 was constructed successfully.Sequencing and basic local alignment search tool （BLAST） analysis showed that there are 6 clones interacting with UL141 encoded protein,of which one clone showed highly homogous with human chromodomain helicase DNA binding protein 3 （CHD3）.Conclusion CHD3 is one of the interaction proteins related to HCMV UL141 in human fetus brain cDNA library,which suggested that UL141 protein may be associated with the immune evasion of the HCMV,interfering with normal cell growth and differentiation.