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

发布时间：2018-05-18 07:06

本文选题：人巨细胞病毒 + UL蛋白　；参考：《吉林大学学报(医学版)》2017年04期

【摘要】：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。
[Abstract]:Objective: To investigate the interaction of human cytomegalovirus (HCMV) UL132 encoding protein by yeast two hybrid system, and to elucidate the possible pathogenesis of cDNA Library in human fetal brain, and to clarify its possible pathogenesis in congenital cytomegalovirus infection. Methods: polymerase chain reaction was used to expand HCMV UL132 fragment, enzyme digestion, and purify the amplified fragment. The yeast decoy expression vector pGBKT7, connecting the HCMV UL132 fragment to the carrier pGBKT7, transformed the linked recombinant pGBKT7-UL132 into yeast AH109, and then transformed the human fetal brain library DNA into yeast AH109 to screen the human fetal brain library protein interacting with the HCMV UL132 encoding protein and sequenced the positive clones and the biological letters. Results: the decoy expression vector pGBKT7-UL132 was successfully constructed, and 2 stripe bands of 800 and 7 500bp were identified by double enzyme digestion; the conversion efficiency was 6.6 * 103 CFU. G-1 after the transformation library plasmid. The color reaction showed that 95 colonies were blue, and 10 clones were interacted with the HCMV UL132 encoded protein, and the Blast results showed 7. The clones are highly homologous with CAML. Conclusion: the human fetal brain cDNA library protein CAML was successfully screened by the yeast two hybrid system, which could interact with the HCMV UL132 encoding protein. It is presumed that the target protein may play an important role in the nervous system damage caused by HCMV infection and the invasion and proliferation of the virus.
【作者单位】：中国医科大学附属盛京医院生物样本库;吉林大学第一医院儿科;
【基金】：国家自然科学基金资助课题(81171581)
【分类号】：R373

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