丙型肝炎病毒NS4A蛋白在胰腺细胞cDNA文库中的结合蛋白基因的筛选

Screening of proteins binding to hepatitis C virus NS4A protein from human pancreas cDNA library

目的:从人胰腺细胞cDNA文库中筛选出与丙型肝炎病毒(HCV)NS4A蛋白存在相关作用并且参与糖、脂类代谢过程的结合蛋白,为研究HCV影响糖、脂类代谢的分子生物学机制提供实验依据。方法:构建pGBKT7-NS4A作为诱饵质粒,转化酵母菌株AH109,用SD/-Trp筛选阳性菌落。配合两种重组酵母菌株AH109与Y187(人胰腺细胞cDNA文库质粒转化),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PUMED进行序列比对。结果:用pGBKT7-HCVNS4A重组质粒作为诱饵,成功从人胰腺cDNA文库中筛选出5种与HCV NS4A蛋白相结合的蛋白基因,包括CDK5R1、弹性蛋白酶3A、胰脂肪酶、KRAS和胆盐刺激酯酶。结论:HCV NS4A蛋白与胰腺文库中的上述5种代谢相关蛋白存在相互作用,从而影响糖代谢过程。

Objective：To screen proteins from human pancreas cDNA library, which interact with the protein coded by hepatitis C virus （HCV） NS4A and relate with the metabolism of glucose or lipid. Methods： The recon- structed plasmid pGBKTT-NS4A was transformed into yeast strain AH109 and identified. The transformed AH109 （pGBKT7-NS4A） mated with Y187 （plasmid of pancreatic cDNA library）. The diploid yeast cells were plated on nutrient deficiency medium containing X-a-gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E. coli DH5a. The plasmids in DHSa were extracted, sequenced and blasted. Results： Five proteins interacting with HCV NS4A were found, including CDK5R1, elastase 3A, pancreatic lipase, KRAS and bile salt-stimulated esterase. Conclusions：The five pancreatic proteins related with metabolisms of glucose and lipid could interact with HCV NS4A, thus disturbing the metabolism of glucose or lipid.