Cloning and Expression of Chinese Duck Interferon-gamma Gene.

ABSTRACT

The efficacy of cytokine therapy has been demonstrated in several viral diseases. Interferon-gamma is a cytokine that has potent antiviral property and immunomodulatory activity. To investigate the role of IFN-gamma in viral clearance during natural infection and to define the antiviral mechanism, DHBV-infected ducks was used as an animal model. To clone, express, and develop the method of quantifying DuIFN-gamma gene transcription and expression, DuIFN -gamma cDNA was amplified by RT-PCR from PHA stimulated duck PBMC. Recombinant plasmid expressing DuIFN-gamma was used to transfect COS-7, and the cell culture supernatant was analyzed by CPE inhibitory assay and MTT methods to determine the antiviral titer of IFN-gamma. The GST-DuIFN-gamma fusion protein was expressed in E.coli and purified using the GST sepharose 4B. Results indicated that the supernatant collected from COS-7 cells transfected with DuIFN-gamma cDNA was able to prevent duck fibroblasts from VSV induced CPE in a dose dependent manner. An anti-DuIFN-gamma antibody neutralized this antiviral activity.