[Cloning of genes transactivated by nonstructural protein 4A of hepatitis C virus.]

To construct a subtractive cDNA library of genes transactivated by NS4A protein of hepatitis C virus with suppression subtractive hybridization technique (SSH).

The mRNA was isolated from Hep G2 cells transfected pcDNA3.1(-)-NS4A and pcDNA3.1(-) empty vector, respectively, then the cDNA was synthesized. SSH method was employed to analyze the differentially expressed RNA sequence between the two groups. The twice enriched PCR products were subcloned into T/A vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain JM109. The cDNA was sequenced and analyzed in comparison with those in GenBank with Blast search after PCR.

The amplified library contained 36 positive clones. Colony PCR showed that 36 clones contained 200-1000 bp inserts. Sequence analysis was performed in 25 clones, and the full length sequences were obtained with bioinformatics method. Altogether 20 kinds of coding sequences were achieved, which consisted of 18 kinds of known and 2 kinds of unknown ones. The obtained sequences may be target genes transactivated by NS4A protein of HCV, among which some genes coding for proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development.

A subtractive library of genes transactivated by NS4A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins.