胰腺细胞中与HCV NS2蛋白相互作用蛋白中代谢相关基因的筛选

Screening of proteins bindingto hepatitis C virus NS2 proteinfrom a human pancreas cDNA library

目的:研究人胰腺细胞中与丙型肝炎病毒(HCV)包膜糖蛋白NS2相互作用的结合蛋白中的代谢相关基因.方法:扩增人胰腺细胞cDNA文库并进行纯化鉴定,将文库质粒转化酵母菌株Y187.诱饵质粒pGBKT7-NS2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4缺培养基(SD/-Trp/-Leu/-His/-Ade)和铺有X-α-gal的4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pGB-KT7-HCVNS2重组质粒,筛选出7种与HCVNS2蛋白相结合的蛋白基因.结论:筛选出的与HCVNS2蛋白结合的部分人胰腺细胞蛋白基因与糖、脂类代谢密切相关.

AIM： To screen proteins interacting with hepatitis C virus （HCV） NS2 protein from a human pancreas cDNA library.METHODS： A human pancreas cDNA library was amplified,purified and identified.The purified library plasmid was transformed into yeast strain Y187.The bait plasmid pGBKT7NS2 was transformed into yeast strain AH109 and screened on SD/-Trp agar plates.The transformed AH109 strain was then mated with Y187 strain that contained the library plasmid.The diploid yeast cells were selected on SD/-Trp/Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade agar plates containing X-α-gal.The plasmid in diploid yeast cells was extracted and electrotransformed into E.coli DH5α strain for plasmid preparation,sequencing and sequence blasting.RESULTS： A human pancreas cDNA library was constructed successfully.The reconstructed bait plasmid （pGBKT7-HCV NS2） was successfully transformed into yeast strain AH109.Seven proteins interacting with the HCV NS2 protein were identified.CONCLUSION： Seven proteins interacting with the HCV NS2 protein is identified from a human pancreas cDNA library,some of which may be related with glucose and lipid metabolism.