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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 the protein interacting with human cytomegalovirus (HCMV) UL / b’UL133 protein by yeast two-hybrid system in order to study the relationship between UL / b ’ The biological function of the encoded protein, revealing the pathogenesis of HCMV congenital infection lay the foundation. Methods Specific primers were designed. Nde Ⅰ and BamH Ⅰ restriction enzyme recognition sites were introduced upstream and downstream of the primers respectively. The UL133 gene fragment of H strain of HCMV clinical isolates was amplified by PCR. The amplified product was used simultaneously with the vector pGBKT7 Nde Ⅰ and BamH Ⅰ double digestion, purified, the UL133 fragment inserted into pGBKT7 vector to construct bait plasmid pGBKT7-UL133, and sequencing analysis. Using the small amount of lithium acetate conversion method, the correct bait plasmid pGBKT7-UL133 was transformed into AH109 yeast competent cells, and then the transformed bacterial cells were coated on tryptophan-deficient medium (-Trp) Positive colonies. Then, the human fetal brain c ;; DOI: 10.3969 / j.issn.NA library plasmid was transformed into AH109 strain containing pGBKT7-UL133 plasmid and cultured in four deficient medium (-Ade / -His / -Leu / -Trp) , The color reaction was carried out on the X-Gal-coated filter paper, the positive blue yeast was screened and the plasmid was electroporated into E. coli of TG1 and identified by PCR, Library plasmid and turned back into a yeast strain containing the bait plasmid pGBKT7-UL133 for gyratory verification. The positive clones screened were sequenced and analyzed by bioinformatics. Results The bait plasmid pGBKT7-UL133 for yeast two-hybrid screening was successfully constructed and the yeast strain containing the bait plasmid was grown on -Trp. Transformed yeast strain AH109 with the human fetal brain c ;; DOI: 10.3969 / j.issn.NA library and the bait plasmid pGBKT7-UL133, 28 yeast colonies were observed in the four missing culture medium. Through the chromogenic reaction, Four positive clones were obtained by transformation and rotary yeast cell screening. One of these genes was found to encode a human reduced nicotinamide adenine dinucleotide phosphate (NAI; DOI: 10.3969 / j.issn.PH) -dependent oxidoreductase 1 by sequence alignment. Conclusion The yeast two-hybrid system was successfully used to screen out the protein -NA interacting with the UL133 coding protein in UL / b ’region of HCMV. DOI: 10.3969 / j.issn.PH, suggesting that the UL133 protein may play an important role in increasing virus virulence and infectivity, Interfere with cell signal transduction and cell growth, division, apoptosis play an important role.