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BACKGROUND:Interferon-alpha(IFN-α)is an important cytokine with multiple functions,but the target genes transactivated by IFN-αremain largely unknown.A study of such genes will help to understand the mechanism of function of IFN-α.To isolate the gene transcripts specifically upregulated by IFN-αin HepG2 cells,we conducted suppressive subtractive hybridization(SSH) analysis. METHODS:SSH was used to analyze the target genes transactivated by recombinant IFN-αprotein,and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-α(rIFN-α,2000 IU/ml)for 16 hours as tester,and cells not treated with rIFN-αas driver.The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected,sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS:The subtractive cDNA library of genes upregulated by IFN-αwas constructed successfully. rIFN-αupregulated the expression of the RAN bindingprotein 5(RANBP5),NADH dehydrogenase,exosome component 3(EXOSC3),zinc finger RNA binding protein, Dickkopf homolog 1(DKK1)and acetyl-coenzyme A acetyltransferase 2(ACAT2). CONCLUSIONS:These results suggest that rIFN-αcan upregulate the expression of important genes to exert its functions,and provide new clues for discovering the molecular mechanisms of action of IFN-α.
BACKGROUND: Interferon-alpha (IFN-α) is an important cytokine with multiple functions, but the target genes transactivated by IFN-αremain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-α.To isolate the gene transcripts specifically upregulated by IFN-αin HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-α protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-α (rIFN-α, 2000 IU / ml) for 16 hours as tester, and cells not treated with rIFN-αas driver.The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. ibrary of genes upregulated by IFN-αwas constructed successfully. rIFN-α regulatedregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-αcan upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-α.