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

作　　者：温少芳[1] 张锦前[1] 孙荣华[1] 李卓[1] 高萍[1] 王琦[1] 刘顺爱[1] 成军[1] 

机构地区：[1]首都医科大学附属北京地坛医院传染病研究所,100015

出　　处：《中华临床医师杂志（电子版）》2011年第9期2523-2526,共4页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学基金(30600524)

摘　　要：目的从人胰腺细胞cDNA文库中,采用酵母双杂交方法筛选HCV NS4B包膜蛋白的相互作用蛋白。方法人胰腺细胞cDNA文库先进行扩增,随后纯化及鉴定,然后将鉴定好的人胰腺细胞cDNA文库质粒转化入酵母菌株Y187。构建pGBKT7-NS4B作为诱饵质粒,随之转化酵母菌株AH109,用色氨酸缺陷型培养基(SD/-Trp)筛选阳性菌落。配合两种重组酵母菌株AH109与Y187,用四缺培养基及X-α-gal筛选蓝色酵母菌落,提取相应质粒,电转化感受态菌DH5α后再提取质粒,测序仪测序,结果使用PUBMED进行序列比对。结果成功构建pGBKT7-HCV NS4B重组质粒,并从人胰腺cDNA文库中筛选出7种与HCV NS4B蛋白相结合的蛋白基因,包括CDK5RAP3、辅脂肪酶、胰石蛋白、凝乳蛋白、弹性蛋白酶2A、糜蛋白酶和胆盐刺激酯酶。结论 HCV NS4B蛋白可能通过与所筛选出前述蛋白中参与代谢过程的相关蛋白结合,影响糖、脂类代谢过程。Objective To screen proteins from human pancreas cDNA library,which interact with the protein coded by hepatitis C virus (HCV) NS4B.Methods The reconstructed plasmid pGBKT7-NS4B was transformed into yeast strain AH109 and identified.The transformed AH109 (pGBKT7-NS4B) mated with Y187 (plasmids of pancreatic cDNA library).The diploid yeast cells were plated on nutrient deficiency medium containing X-α-gal for selecting.The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5α.The plasmids in DH5α were extracted,sequenced and blasted.Results Seven proteins interacting with HCV NS4B were found,such as CDK5RAP3,colipase,pancreatic stone protein,lactoprotein,elastase2A,chymotrypsin and bile salt-stimulated esterase.Conclusions Some of the eight pancreatic proteins may be related with metabolisms of glucose and lipid.

关 键 词：肝炎病毒属 基因文库 胰腺 载体蛋白质类 双杂交系统技术 NS4B 

分 类 号：R346[医药卫生—基础医学]