کلون سازی فعال کننده پلاسمینوژن انسانی نوترکیب به روش RT-PCR

Cloning the gene , responsible for the production of human tissue –type plasminogen activator (ht-PA) protein.t-PA is a 527 amino acid glycopeptide whit thrombolytic activity.It binds to fibrin by it's amin end and facilate fibrinolysis by activating plasmin production from plasminogen. Total RNA was extracted from Fibrosarcoma cell line (HT-1080). RT-PCR was employed to produce t-PA cDNA and was subsequently cloned into pCR2.1 vector and XL1-B cells were transformed.The cloned gene was subjected to restriction endonuclease mapping to confirm cloning of the gene. following RT-PCR reaction, RCR product was cloned into pCR2.1 vector and positive colonies in blue-white screening was picked.DNA was extracted and by using restriction enzymes, the cDNA was confirmed.The gene was cut and subcloned into pcDNA3.1 expretion vector.The final sequence was confirmed using DNA sequencing. The cloning of human tPA cDNA in an expression vector is the first step in recombinant production of this protein. Since the production of biological proteins using biotechnology, proved to be safe from contamination during purification step from tissue. We hope that cloning of human tPA will open the path to recombinant production of this valuable drug.