Objectives: To identify and clone human genes transactivated by HCV F protein by constructing a cDNA subtractive library using the suppression subtractive hybridization technique.
Methods: Suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV F protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-F or with pcDNA3.1(-) empty vector as a control, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 or adaptor 2. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 alpha. The cDNA was sequenced and analyzed in GenBank with blast search after PCR.
Results: The subtractive library of genes transactivated by HCV F protein was constructed successfully. The amplified library contains 71 positive clones. Colony PCR shows that 56 clones contain 200-1000 bp inserts. Sequence analysis was performed on 28 clones randomly, and the full length sequences were obtained with using the bioinformatics method. Altogether 19 coding sequences were obtained, consisting of 17 known and 2 unknown.
Conclusions: The obtained sequences may be target genes transactivated by HCV F protein, and some gene coding proteins are those involved in cell cycle regulation, metabolism, and cell apoptosis.