Study of T1 Tobacco Transplastomic Plants Containing Human Gamma Interferon Gene (hIFNg)

In the recent decades the progress in plant biotechnology, especially in the field of molecular farming, has made it possible to produce a wide range of recombinant protein using plants as a novel production system and bioreactor. human gamma interferon is one of the valuable pharmaceutical proteins which has found wide medical applications in diagnosis and therapeutic. Cloning of human gamma interferon gene associated with spectinomycinstreptomycin antibiotic resistance gene using chloroplast pKCZ vector has been performed at agricultural biotechnology laboratory of Tarbiat Modares University and has been transferred into the tabacco chloroplast genome with biolistic method. The purpose of this study is to determine gamma interferon gene expression stability in T1 tobacco transplastomic plants. plants, which are grown on selective media, were analysed at different levels of (DNA, RNA and protein) to confirm integration and expression of the gene into the tobacco chloroplast. Analysis of plants was performed at DNA level by using of PCR and specific primers for human gamma interferon gene, results confirmed integration of this gene into the chloroplast genome. In addition, transcription of target gene was confirmed by RT-PCR. In order to study expression of target gene at protein level SDS-PAGE, Dot blotting and Western-blot were also done and the obtained results show target gene expression at protein level. Finally, ELISA test was done to determine quantity of gamma interferon gene expression. Results indicated that the highest gamma interferon level in transplastomic plant is up to 0.2% of total soluble protein.