Cloning of the truncated Human tPA Gene in Chloroplast vector and regeneration of transplastomic tobacco plants

Plants are suitable replacements for current biodrugs and recombinant protein expression systems such as transgenic animals or microbial systems. As the plant plastid genome is highly polyploid, the transformation of chloroplast permits the introduction of thousands of copies of foreign gene per plant cell and generates extraordinarily high levels of recombinant protein. Cardiovascular diseases are the second one leading cause of human mortality after cancer. Human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of body such as brain and heart blood vessels. The truncated form of tissue plasminogen activator (K2S) has longer plasma half life, better diffusion into the clot and higher fibrinolytic activity. In this study, in order to introduction of K2S gene in tobacco chloroplast, after construct design, the interest gene was ligated to pKCZ vector and cloned in E. coli. Following the successfully shooting of the K2S-containing vector (pKCZK2S) to leaf explants using the biolistic delivery procedure, the explants were selected on selection medium containing 500 mgL-1 spectinomycine antibiotic. After regeneration of grown shoots on selective medium, in order to achieve homoplasmy, fourth rounds of selection and regeneration in presence of spectinomycine antibiotic were performed. The presence, site-specific integration, expression of the K2S gene and homoplasmy in transplastomic plants were confirmed by PCR, RT-PCR and Southern blotting methods.