Eukaryotic expression of cis-inserted double copies of DuIFN-γ gene

Objective:In this study ,duck interferon-gamma(DuIFN-γ) gene has been used as target gene to investigate the effect of copy number of cloned foreign gene on the expression of target protein under control of a single promoter .Methods:The 1.4kb size of DuIFN-γ cDNA was cleaved from plasmid pGEM-DuIFN-γ with EcoRI and subcloned into the EcoRI site of pcDNA1.1/Amp vector.The copy number and orientation of insert in recombinant plasmids were characterised by restriction enzyme analysis and transfected into COS-7 cells for expression with FuGENE6 transfection reagent.The biological activity of DuIFN-γ in cultured supernatant was assayed with Griess reagent for its capacity of activating chicken macrophage cell line HD11 to secrete nitric oxide. The recombinant plasmids with one or two copies of forward inserts were identified with restriction enzyme analysis.Results: The results of DuIFN-γ biological assay showed that 50% of maximal DuIFN-γ activity was reserved after 1∶160 dilution of cultured supernatant transfected by recombinant plasmid with single copy of DuIFN-γ cDNA,the cultured supernatant of transfected COS-7 cells remained half of the maximal activity of DuIFN-γ following 1∶320 dilution.In addition ,the DuIFN-γ was still active after 10240 fold dilution for plasmid wth double copies of DuIFN-γ genes ,but inactive after 1280 fold dilution for plasmid with single copy of DuIFN-γ gene.Conclusion:By sharing the same promoter ,the level of target protein (DuIFN-γ) expressed by recombinant plasmid with two copies of forward insert was obviously enhanced in eukaryotic cells.