Heterologous expression of synthetic chicken IFN-γ in transgenic tobacco plants

Abstract

To develop a plant expression system for the production of the chicken interferon gamma (ChIFN-γ) oral vaccine adjuvant, we investigated whether the ChIFN-γ protein can be expressed in tobacco plants. The coding sequence of the ChIFN-γ gene was optimized by modification of codon usage to that of tobacco plant genes. A synthetic ChIFN-γ gene was inserted into plasmid pSW-IFNG containing the CaMV35S promoter, NOS terminator, GUS (β-glucuronidase) reporter gene and the nptII resistance gene. The synthetic ChIFN-γ gene, along with an endoplasmic reticulum retention signal (SEKDEL) was introduced into tobacco plants via Agrobacterium-mediated transformation. PCR analysis confirmed the presence of the ChIFN-γ gene in transformants. Histochemical GUS assay of the tobacco leaves revealed stable integration of ChIFN-γ into the genome. RT-PCR analysis revealed the presence of ChIFN-γ-specific transcript. Bands of approximately 30 and 40 kDa were detected by Western analysis of transgenic tobacco leaves using an anti-chicken IFN-γ antibody. Furthermore, quantitative ELISA detected ChIFN-γ protein, suggesting that tobacco leaves expressed ChIFN-γ of up to 10 to 20 μg/g fresh leaf weight, or 0.02 to 0.04% of total soluble protein. ChIFN-γ present in tobacco extracts possessed antiviral activity (4.8×105 IU/mg in vitro). The present results indicate that the ChIFN-γ introduced into tobacco plant was correctly transcribed and translated in plant. High-level expression of biologically active ChIFN-γ will allow further studies of oral administration and therapeutic effects of this cytokine. © 2009 Versita Warsaw and Springer-Verlag Berlin Heidelberg.