Transactivating effect of hepatitis C virus core protein: A suppression subtractive hybridization study

作     者：MinLiu YanLiu ]unCheng Shu-LinZhang LinWang QingShao JianZhang QianYang 

作者机构：DepartmentofInfectiousDiseasesTheFirstHospitalofXi'anJiaotongUniversityXi'an710061ShaanxiProvinceChina GeneTherapyResearchCenterInstituteofInfectiousDiseases302HospitalofPLABeijing100039China GeneTherapyResearchCenterInstituteofInfectiousDiseases302HospitalofPLABeijing100039China GeneTherapyResearchCenterInstituteofInfectiousDiseases.302HospitalofPLABeijing100039China DepartmentofInfectiousDiseasesTheFirstHospitalofXi'anJiaotongUniversityXi'an710061ShaanxiProvinceChina 

出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)

年 卷 期：2004年第10卷第12期

页      面：1746-1749页

核心收录：

学科分类：1004[医学-公共卫生与预防医学(可授医学、理学学位)] 1002[医学-临床医学] 100401[医学-流行病与卫生统计学] 10[医学] 

基　　金：Supported by the National Natural Science Foundation of China No.39970674 

主　　题：HCV 丙型肝炎病毒 核心蛋白质 抑制作用 杂交作用 肝脏疾病 

摘      要：AIM: To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein. METHODS: pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BarnHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cell swere transiently transfected with pcDNA3.1(-)-core using Upofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coil strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR). RESULTS: The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of β-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR showed that 213 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by Genl3ank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein. CONCLUSION: The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced. Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.