Cloning of genes transactivated by nonstructural protein 5B of hepatitis C virus

Objective: To construct a subtractive cDNA library of genes transactivated by NS5B protein of hepatitis C virus with suppression subtractive hybridization technique（SSH）.Methods: The mRNA was isolated from HepG2 cells transfected pcDNA3.1（-）-NS5B 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 cDNA was sequenced and analyzed in GenBank with Blast search after the PCR amplification of the library.Results: The amplified library contains 35 positive clones.（Colony） PCR shows that 26 clones contain 200-1000 bp inserts.Sequence analysis was performed in 26 clones,and the full length sequences were obtained with bioinformatics method.Altogether 17 kinds of coding sequences were achieved,which consisted of 16 kinds of known and one unknown gene.The obtained sequences may be target genes transactivated by NS5B protein of HCV,（among） which some genes coding proteins involved in cell cycle regulation,cell signal transduction pathway and tumour develo（pment）.Conclusions: A subtractive library of genes transactivated by NS5B protein of HCV was constructed successfully,which brought some new clues for studying the biological functions and pathogenesis of the viral proteins.hepatitis C-like virus;viral nonstructural proteins;transactivation（genetics）