与人巨细胞病毒UL132编码蛋白相互作用蛋白的初步筛选和鉴定

Screening and identification of proteins interacting with HCMV UL132 protein

目的:探讨应用酵母双杂交系统筛选出与人巨细胞病毒(HCMV)UL132编码蛋白相互作用的人胎脑cDNA文库组织蛋白,阐明其在先天性巨细胞病毒感染中可能的致病机制。方法:采用聚合酶链式反应扩增HCMV UL132片段,酶切、纯化扩增的目的片段及酵母诱饵表达载体pGBKT7,连接HCMV UL132片段到载体pGBKT7上,将连接后的重组体pGBKT7-UL132转化到酵母菌AH109中,再将人胎脑文库DNA也转化到酵母AH109中,筛选与HCMV UL132编码蛋白相互作用的人胎脑文库蛋白,并对阳性克隆进行测序和生物信息学分析。结果:成功构建诱饵表达载体pGBKT7-UL132,双酶切鉴定可见800及7 500bp 2条目的条带;转化文库质粒后计算转化效率为6.6×103 cfu·μg-1,显色反应显示有95个菌落呈蓝色,最终确认10个克隆与HCMV UL132编码蛋白相互作用,Blast结果显示7个克隆与CAML高度同源。结论:利用酵母双杂交系统成功筛选出与HCMV UL132编码蛋白相互作用的人胎脑cDNA文库蛋白CAML,推测该目的蛋白可能在HCMV感染所致神经系统损伤、病毒的入侵和增殖过程中发挥重要作用。

Objective： To screen the proteins interacting with the human cytomegalovirus （HCMV） UL132 protein from the human fetus brain cDNA library by using Yeast Two-Hybrid System, and to elucidate the possible mechanism of UL132 protein in congenital cytomegalovirus infection. Methods.. The HCMV UL132 fragment was amplified by polymerase chain reaction, the amplified HCMV UL132 fragment and expression vector pGBKT7 were digested and purified, and the HCMV UL132 fragment was linked to the vector pGBKTT. The pGBKTT-UL132 was constructed and transformed to yeast AH109, then the Human Fetal Brain DNA Library DNA was transformed into AH109 yeast. Using HCMV UL132 as abait, a human fetus brain cDNA was screened and the proteins interacting with UL132 protein were searched, the positive clone was sequenced and analyzed by bioinformaties methods. Results： The bait expression vector pGBKT7-UL132 was successfully constructed. The results of double enzyme digestion showed that there were two visible bands of 800 and 7 000 bp, respectively. After transformation of library plasmid, the transformation efficiency was calculated, and the transformation efficiency was 6.6×10^3 cfu ·μg 1. There were 95 blue clones by X -gal coloration reactionsequencing and there were 10 clones interacting with the protein encoded by UL141 protein. The BLAST analysis showed that 7 of them were highly homologous with CAML. Conclusion; CAML might be one interaction protein with HCMV UL132 in Human Fetus Brain cDNA Library, suggesting that the interaction may be associated with the invasion and proliferation of the HCMV.