人胰腺细胞cDNA文库中丙型肝炎病毒NS4B结合蛋白基因的筛选

Screening of proteins binding to hepatitis C virus NS4B protein from human pancreas cDNA library

目的应用酵母双杂交技术筛选人胰腺细胞cDNA文库中与HCV包膜糖蛋白NS4B相互作用的结合蛋白的编码基因。方法扩增人胰腺细胞cDNA文库进行纯化鉴定,并将文库质粒转化酵母菌株Y187。诱饵质粒pGBKT7-NS4B转化酵母菌株AH109,在色氨酸缺陷型培养基(SD/-Trp)上筛选阳性菌落。应用酵母双杂交系统3将阳性重组AH109菌株与重组酵母菌株Y187进行配合,在四缺培养基(SD/-Trp/-Leu/-H is/-Ade)和铺有X-α-gal的四缺培养基上进行筛选,提取蓝色酵母菌落质粒,电转化大肠埃希菌DH5α后再提取质粒测序。测序结果进行序列比对。结果成功构建人胰腺cDNA文库以及pGBKT7-HCV NS4B重组质粒,筛选出7种与HCV NS4B蛋白相结合的蛋白基因。结论筛选出的与HCV NS4B蛋白结合的人胰腺细胞蛋白基因中,部分与2型糖尿病、肝脏脂肪变性密切相关。

Objective To screen gene encoding of pancreatic proteins from human pancreas cDNA library,which interact with hepatitis C virus（HCV） NS4B protein.Methods The human pancreas cDNA library was amplified,purified and evaluated.The purified library plasmids were transformed into yeast strain Y187.The reconstructed plasmid pGBKT7-NS4B was transformed into yeast strain AH109 and the positive colonies were screened on the nutrient deficiency medium SD/-Trp.The transformed AH109 mated with Y187 that contained the library plasmids via the yeast two hybridization system 3.The diploid yeast cells were placed on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-α-gal for selecting.The Blue yeast colony plasmids were extracted and eleetrotransformed into E.coli DH5α.The plasmids in DH5α were extracted,sequenced and blasted.Results The human pancreas cDNA library was constructed successfully.The reconstructed bait plasmid（pGBKT7-HCV NS4B） was transformed into yeast cells AH109 successfully.Seven proteins interacting with HCV NS4B were found.Conclusion Some of the seven pancreatic proteins may be related with metabolisms of glucose and lipid.