筛选与分析与人巨细胞病毒UL133编码蛋白相互作用的蛋白

Screening and Analysis of Proteins Interacting with Human Cytomegalovirus UL133 Protein

目的应用酵母双杂交技术筛选人胎脑c;;DOI:10.3969/j.issn.NA文库中与人巨细胞病毒(HCMV)UL/b′UL133编码蛋白相互作用的蛋白,为研究UL/b′编码蛋白的生物学功能,揭示HCMV先天感染的致病机制奠定基础。方法设计特异性引物,在引物上下游分别引入NdeⅠ和BamHⅠ限制性内切酶识别位点,采用PCR技术扩增HCMV临床分离株H株的UL133基因片段,将其扩增产物与载体pGBKT7同时使用NdeⅠ和BamHⅠ进行双酶切,纯化后,将UL133片段插入到pGBKT7载体上,构建诱饵质粒pGBKT7-UL133,并测序分析。利用醋酸锂小量酵母转化方法 ,将测序正确的诱饵质粒pGBKT7-UL133转化入AH109酵母感受态细胞中,然后将转化的菌液涂布在色氨酸缺陷型培养基(-Trp)上,筛选阳性菌落。再将人胎脑c;;DOI:10.3969/j.issn.NA文库质粒转化到含pGBKT7-UL133质粒的AH109菌株中,在四缺培养基(-Ade/-H is/-Leu/-Trp)中筛选,在铺有X-Gal的滤纸上进行显色反应,提取显蓝色的阳性酵母筛选质粒,运用电穿孔方法将其转化到TG1的大肠杆菌中,并行PCR鉴定,提取大肠杆菌中的文库质粒,并将其回转到含诱饵质粒pGBKT7-UL133的酵母菌株中,进行回转验证。对筛选到的阳性克隆进行测序和生物信息学分析。结果用于酵母双杂交筛选的诱饵质粒pGBKT7-UL133成功构建,含有诱饵质粒的酵母菌株在-Trp上生长。转化有人胎脑c;;DOI:10.3969/j.issn.NA文库和诱饵质粒pGBKT7-UL133的酵母菌AH109,在四缺培养基中观察到有28个酵母菌落生长,通过显色反应、酵母质粒转化及回转酵母细胞筛选得到4个阳性克隆。通过序列比对,发现在这些基因中,其中一个基因编码人类还原型烟酰胺腺嘌呤二核苷酸磷酸(NA;;DOI:10.3969/j.issn.PH)依赖性氧化还原酶1。结论成功应用酵母双杂交系统筛选出与HCMV UL/b′区UL133编码蛋白相互作用的蛋白-NA;;DOI:10.3969/j.issn.PH,提示UL133蛋白可能在增加病毒毒力和传染性,干扰细胞间的信号传导和细胞的生长、分裂、凋亡等方面发挥重要作用。

Objective To screen and identify the proteins interacting with UL/b′ region of the human cytomegalovirus（HCMV） UL133 protein in human fetus brain cDNA library for further in-depth study the pathogenic mechanism of HCMV basic.Methods Special primers with NdeⅠand BamHⅠrestriction sites were designed,the DNA fragment of HCMV UL133 was amplified by polymerase chain reaction from HCMV infected clinical strain H.After UL133 gene and pGBKT7 were verified with restriction endonuclease digestion of NdeⅠand BamHⅠ,UL133 gene was cloned into pGBKT7 vector.Then fragment of recombinant plasmid pGBKT7-UL133 was verified by DNA sequencing.Then right fragment of recombinant plasmid pGBKT7-UL133 was transformed into the yeast cell AH109 by lithium acetate.The transformed yeast cells were plated on nutrient deficiency medium-Trp,then human fetus brain cDNA library was also transformed into the yeast cell AH109 containing the pGBKT7-UL133 plasmid.The yeast cells were plated on nutrient deficiency medium-Ade/-His/-Leu/-Trp and filter paper containing X-Gal for selection.Screening positive clones by yeast plasmid extracting were transformed into E.coli TG1 and transformed into yeast cell one by one,then positive clones were sequenced and analyzed using bioinformatic methods.Results The bait plasmid pGBKT7-UL133 was constructed successfully.The co-lonies containing pGBKT7-UL133 were grown in the-Trp.The positive co-lonies containing pGBKT7-UL133 and cDNA library were 28 colony in-Ade/-His/-Leu/-Trp,sequencing and basic local alignment search tool（BLAST） analysis showed that there were 4 clones interacting with the protein encoded by UL133 protein,of which one clone highly homogous to NADPH.Conclusions HCMV UL133 protein may be interactived with NADPH,UL133 protein may play an important role in the course of HCMV infection,such as signal transmission among cells,growth and apoptosis of cells.