Expression and Purification of Human Interferon Gamma Using a Plant Viral Vector

Authors

1 Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.

2 National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

3 Plant Pathology Department, National Cheng Hsing University, Taichnug, Taiwan.

Abstract

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.
Progress in Biological Sciences
Volume 2, Issue 2 - Serial Number 2
March 2013
Pages 104-115

Files

Share

How to cite

Statistics