expression and purification of human interferon gamma using a plant viral vector

a plant viral vector engineered from an in vivo infectious clone of zucchini yellow mosaic virus(zymv) was used to express the human interferon-gamma (inf-γ) in planta. the inf-γ gene was in frame inserted between the p1 and hc-pro orfs of the zymv vector. the infectious activity of the vector was approved by rubbing the plasmid on chenopodium quino a and observing local lesions. individual lesions were mechanically transferred to the systemic host plant zucchini squash at the stage of cotyledonary leaf. the stability of inf-γ expression was assessed by successive passages of recombinant viruses from infected plant and throughout the period of 35 days after inoculating in a single plant. then, the leaf tissues ofinoculated plant were analyzed for the presence of transgene by rt-pcr and western blot analysis. the recombinant protein was purified using affinity chromatography method. the results showed approximately 1–1.2 mg inf-γ per 100 g tissues were purified from leaves two weeks post inoculation. also, the vector was remarkably stable in squash after six serial passages and 35 days. the procedure provides a convenient and fast method for production of large quantities of pure inf-γin planta. the system also has a potential for production of other proteins of interest in cucurbits to use as immunogen to produce antiserum or use for other purposes.