丙型肝炎病毒非结构蛋白NS4B肝细胞结合蛋白基因的筛选与克隆

Screening and cloning of hepatitis C virus non-structural protein 4B interacting protein gene in hepatocytes

目的筛选并克隆人肝细胞cDNA文库中与丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)相互作用蛋白的基因,明确其具体作用机制。方法应用酵母双杂交系统3,将多聚酶链反应(PCR)法扩增的HCVNS4B基因连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和Xα半乳糖(Xαgal)上进行双重筛选阳性菌落,提取阳性酵母菌落的质粒转化大肠埃希菌,接种在氨苄西林LB平板上,选择生长菌落,提取质粒酶切鉴定,测序并在GenBank中进行生物信息学分析。结果成功克隆出HCVNS4B基因并在酵母细胞中表达,与肝文库配合后选出既能在4缺(SD/TrpLeuAdeHis)培养基又能在铺有Xαgal的4缺培养基上生长,并变成蓝色的真阳性菌落5个,序列分析显示,筛选到的肝细胞蛋白编码基因参与细胞代谢、生物氧化、生长调节等多种生物学过程。结论成功克隆出HCVNS4B蛋白与肝细胞相互作用蛋白,为进一步研究NS4B蛋白的功能,阐明HCV致病的分子生物学机制提供了新线索。

Objective To investigate biological functions of non-structural protein 4B （NS4B） of hepatitis C virus （HCV）, yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4B. Methods HCV NS4B bait plasmid was constructed by ligating the NS4B gene with carrier plasmid pGBKT-/ and transformed into yeast cells AH109 （ type a）. The transformed yeast cells were amplified and mated with yeast cells Y187 （α type） containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His- Ade） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） containing x-α-gal for selecting two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent Escherichia coli and analysed by DNA sequencing and bioinformatics. Results Five genes in eight positive colonies were obtained. There were one NADH dehydrogenase subunit 3, one cytochrome c oxidase subunit Ⅲ, one retinol binding protein 4, one retieulon 3-A （RTN3） and one fibrinogen gamma polypeptide （FGG）. Conclusion Genes of HCV NS4B interacting proteins in hepatocytes were successfully cloned and the results paved the way for studying the biological functions of NS4B and associated proteins.