丙型肝炎病毒核心蛋白的反式激活作用：抑制性消减杂交研究
Transactivating effect of hepatitis C virus core protein: a suppression subtractive hybridization study.
作者信息
Liu Min, Liu Yan, Cheng Jun, Zhang Shu-Lin, Wang Lin, Shao Qing, Zhang Jian, Yang Qian
机构信息
Department of Infectious Diseases, The First Hospital of Xi'an Jiaotong University, Shaanxi Province, China.
出版信息
World J Gastroenterol. 2004 Jun 15;10(12):1746-9. doi: 10.3748/wjg.v10.i12.1746.
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 beta-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 beta-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.
目的
研究丙型肝炎病毒（HCV）核心蛋白的反式激活作用，并筛选被HCV核心蛋白反式激活的基因。
方法
通过将HCV核心基因插入EcoRI/BamHI位点构建含全长HCV核心基因的pcDNA3.1(-)-core。将pcDNA3.1(-)-core与pSV-lacZ共转染HepG2细胞。48小时后，收集细胞并用酶联免疫吸附测定（ELISA）试剂盒检测β-半乳糖苷酶的表达。使用Lipofectamine试剂将pcDNA3.1(-)-core瞬时转染HepG2细胞。收集细胞并分离总mRNA。构建差减cDNA文库并将其构建到pGEM-Teasy载体中。用大肠杆菌JM109菌株扩增文库。聚合酶链反应（PCR）后对cDNA进行测序，并在GenBank中用BLAST搜索进行分析。
结果
在被pcDNA3.1(-)-core转染的HepG2细胞裂解物中可检测到核心mRNA和蛋白。被pcDNA3.1(-)-core转染的HepG2细胞中β-半乳糖苷酶的活性比被对照质粒转染的HepG2细胞高5.4倍。成功构建了被HCV核心蛋白反式激活的基因的差减文库。扩增后的文库包含233个阳性克隆。菌落PCR显示213个克隆含有100 - 1000 bp的插入片段。对63个克隆进行了序列分析。其中6个序列为未知基因。用生物信息学方法获得了全长序列，并被GenBank接受。提示6个新的cDNA序列可能是被HCV核心蛋白反式激活的靶基因。
结论
HCV核心蛋白对SV40早期启动子/增强子有反式激活作用。从cDNA文库中随机选取63个克隆进行测序。使用美国国立生物技术信息中心（NCBI）的BLAST程序分析，其中6个序列为未知基因。其余57个序列与已知基因高度相似。
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