与人巨细胞病毒UL23蛋白相作用的宿主蛋白分子筛选与鉴定

发布时间：2018-01-06 16:18

本文关键词：与人巨细胞病毒UL23蛋白相作用的宿主蛋白分子筛选与鉴定　出处：《暨南大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的：利用酵母双杂系统来筛选与人巨细胞病毒蛋白pUL23相互作用的宿主蛋白,并通过GST-pull down和免疫共沉淀技术对蛋白之间的相互作用进行确认,为进一步研究病毒蛋白pUL23的功能奠定基础。方法：1)酵母双杂交试验：以BAC-HCMV TOWNE为模板扩增UL23基因的ORF序列,构建pGBKT7-UL23诱饵质粒,并扩增人胚肾cDNA文库,通过共转化酵母AH109进行酵母双杂交试验,挑选长势良好的克隆100个进行β-半乳糖苷酶活性检测,确定其为阳性克隆,抽提阳性克隆中的文库质粒,电转化至大肠杆菌E.coli DH5α中扩增,PCR文库插入片断,并进行HaeⅢ酶切去重复,然后将含有非重复片段的克隆送进行测序,对测序结果在GenBank上进行同源性搜索和序列比对分析。2)GST-pull down试验：以人胚肾cDNA文库为模板,通过PCR方法扩增人DNAJB6 [DnaJ (Hsp40) homolog, subfamily B, member 6(DNAJB6)]基因CDS,插入pGEX4-T-1载体中,IPTG诱导产生GST-DNAJB6融合蛋白；构建pcDNA3.1(-)-HA-UL23质粒,通过Cos-7细胞瞬时表达,进行GST-pull down试验检测pUL23蛋白与人DNAJB6蛋白间的相互作用。3)免疫共沉淀试验：构建pcDNA3.1(+)-HA-DNAJB6和pcDNA3.1(+)-Flag-UL23质粒,共转染Cos-7细胞,采用免疫共沉淀试验进一步验证pUL23蛋白与人DNAJB6蛋白间的相互作用。结果：酵母双杂交试验得到79个阳性克隆,其中一个克隆为人DNAJB6 [DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6)]基因。GST-pull down实验和免疫共沉淀试验进一步验证pUL23能与人DNAJB6蛋白相互作用。结论：本课题运用酵母双杂交系统筛选出与HCMV有相互作用的宿主蛋白DNAJB6,并通过GST-pull down和免疫共沉淀试验确认其相互作用,试验结果将为进一步研究pUL23蛋白在HCMV生活周期中的作用机制提供线索与依据。
[Abstract]:Objective : To screen the host protein interacting with human cytomegalovirus protein pUL23 by using yeast two - hybrid system , and confirm the interaction between proteins by GST - pull down and immune co - precipitation technique . Methods : 1 ) Yeast two - hybrid assay : Using BAC - HCMV TOWNE as template to amplify the ORF sequence of UL23 gene , construct pGBKT7 - UL23 bait plasmid , and amplify human embryo kidney cDNA library . The recombinant plasmid pcDNA3.1 ( + ) - HA - DNAJB6 and pcDNA3.1 ( + ) - Flag - UL23 plasmid were inserted into pGEX4 - T - 1 vector . Results : Seventy - nine positive clones were obtained from yeast two - hybrid test . One of them was the human DNA JB6 - DnaJ ( Hsp40 ) gene . The GST - pull down experiment and the immuno - co - precipitation test further verified that pUL23 could interact with the human DNA JB6 protein . Conclusion : The yeast two - hybrid system is used to screen the host protein DNAJB6 which interacts with HCMV , and its interaction is confirmed by GST - pull down and immune co - precipitation test . The experimental results will provide clues and basis for further study on the mechanism of pUL23 protein in HCMV life cycle .

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373

【引证文献】