Cloning of genes transactivated by hepatitis C virus E2 protein by suppression subtractive hybridization technique

Objective To construct a subtractive cDNA library of genes transactivated by hepatitis C virus (HCV) E2 protein using suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivating function.Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)_E2 and pcDNA3.1(-) empty vector, respectively, then cDNA was synthesized.After restriction enzyme RsaI digestion, small size cDNAs were obtained.Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor.After tester cDNA was hybridized with driver cDNA twice,and underwent nested polymerase chain reaction (PCR) twice and then the amplified cDNA fragments were subcloned into T/A plasmid vectors to set up the subtractive cDNA library.Amplification of the library was carried out with E.coli strain JM109.The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.Results The subtractive cDNA library of genes transactivated by HCV E2 was constructed successfully.The amplified library contains 78 positive clones.Colony PCR shows that these clones contain 100～1 000bp inserts.Thirty_eight clones were analyzed by sequencing and bioinformatics.Thirty_five known genes and three genes with unknown function were obtained.Conclusion A subtractive cDNA library of genes transactivated by HCV E2 protein using SSH technique was constructed successfully.The obtained sequences may be target genes transactivated by HCV E2 protein, which brought some new clues for studying the biological functions of HCV E2 protein.