Viral Hepatitis
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 15, 2004; 10(12): 1746-1749
Published online Jun 15, 2004. doi: 10.3748/wjg.v10.i12.1746
Transactivating effect of hepatitis C virus core protein: A suppression subtractive hybridization study
Min Liu, Yan Liu, Jun Cheng, Shu-Lin Zhang, Lin Wang, Qing Shao, Jian Zhang, Qian Yang
Min Liu, Shu-Lin Zhang, Qian Yang, Department of Infectious Diseases, The First Hospital of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Yan Liu, Jun Cheng, Lin Wang, Qing Shao, Jian Zhang, Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, Beijing 100039, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No.39970674
Correspondence to: Dr. Jun Cheng, Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, 100 Xisihuanzhong Road, Beijing 100039, China. cj@genetherapy.com.cn
Telephone: +86-10-66933392 Fax: +86-10-63801283
Received: November 21, 2003
Revised: December 22, 2003
Accepted: December 29, 2003
Published online: June 15, 2004
Abstract

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/BamHI 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 cells were transiently transfected with pcDNA3.1(-)-core using Lipofectamine 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. coli 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 GenBank. 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.

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