Cloning of genes transactivated by hepatitis B virus complete S protein by suppression subtractive hybridization technique

Objective To construct a subtractive cDNA library of genes transactivated by hepatitis B virus (HBV) complete surface 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 by pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sizes cDNA 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, underwent nested polymerase chain reaction (PCR) twice, and then was 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.Results The cDNA was sequenced and analyzed. Genes transactivated by HBV complete S was constructed successfully. The amplified library contained 86 positive clones. Colony PCR showed that these clones contained 100-1000 bp inserts. Thirty-five clones were analyzed by sequencing and bioinformatics. Thirty-three known genes and two genes with unknown function were obtained. Conclusions A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique was constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protin, which brought some new clues for studying the biological functions of HBV complete S protin.