论文部分内容阅读
目的:用酵母双杂交技术筛选白细胞中与丙型肝炎病毒(HCV)非结构蛋白3(NS3)反式调节靶基因编码产物NS3TP6 蛋白结合蛋白的基因. 方法:用多聚酶链反应(PCR)法扩增HCV NS3TP6蛋白基因,连接入酵母表达载体pGBKT-7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人白细胞文库质粒的酵母细胞Y187进行配合,在营养缺陷型培养基上进行双重筛选阳性菌落,增菌后提取质粒,转化入大肠杆菌,提取质粒并测序,进行生物信息学分析. 结果:成功克隆出HCV NS3TP6蛋白基因并在酵母细胞中表达,配合后选出在四缺(SD/-Trp-Leu-Ade-His)培养基和铺有X-α-半乳糖(X-α-gal)的四缺培养基上均能生长并变成蓝色的真阳性菌落共7个,其中3个人类白细胞CD14抗原,1个人类免疫球蛋白入轻链,3个人类推定蛋白基因(AC124014,AC097504,AC023785). 结论:成功克隆出NS3TP6蛋白的结合蛋白,为进一步研究HCV的作用提供了新线索.
OBJECTIVE: To screen the genes encoding the NS3TP6 protein binding protein of the target gene of non-structural protein 3 (NS3) transactivated by hepatitis C virus (HCV) in leukocytes by yeast two-hybrid technique.Methods: Polymerase chain reaction The HCV NS3TP6 protein gene was added and ligated into the yeast expression vector pGBKT-7 to construct a bait plasmid. The bait plasmid was transformed into the AH109 yeast cell and then transformed into the yeast cell Y187 transformed with the human leukocyte library plasmid. The positive colonies were screened by double screening, and the plasmids were extracted after enrichment, transformed into E.coli, and the plasmids were extracted and sequenced for bioinformatics analysis.Results: HCV NS3TP6 protein gene was successfully cloned and expressed in yeast cells, True-positive colonies that grew on both SD / -Trp-Leu-Ade-His and X-α-galactose (X-α-gal) A total of 7 human leucocyte CD14 antigens, 1 human immunoglobulin into the light chain and 3 human putative protein genes (AC124014, AC097504, AC023785) .Conclusion: The cloned NS3TP6 protein was successfully cloned for further study The role of HCV Provide new clues.