Cloning and Analysis of Expressed Genes Transactivated by HCVFBP2 Protein by Suppression Subtractive Hybridization

Objective:To construct a subtractive cDNA library of genes transactivated by homo sapiens HCVFBP2 using suppression subtractive hybridization(SSH)technique and to clone genes associated with its transactivating function.Methods:The mRNA was isolated from HepG2 cells transfected pcDNA 3.1(-)-FBP2 and pcDNA 3.1(-) empty vector,respectively,to synthesize cDNA.After restriction enzyme Rsa Ⅰ digestion,small sizes cDNA were obtained.Then tester cDNA was subdivided into two portions and each ligated with different cDNA adaptor.After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction(PCR)twice and then was subcloned into pGEM-Teasy vectors to set up the subtractive cDNA library.Amplification of the library was carried out with E.coli strain DH5α.The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.Results:The subtractive cDNA library of genes transactivated by HCVFBP2 was constructed successfully.From the resulting collection of clones,sequence information was obtained for a total of 30 distinct transcripts.Of these,28 were found to correspong to known genes,2 matched expressed sequence tags in public databases.Conclusion:A subtractive cDNA library of genes transactivated by HCVFBP2 using SSH technique was constructed successfully.The obtianed sequences may be target genes transactivated by HCVFBP2, which brought some new clues for studying the biological functions of HCVFBP2.