Original Articles
Hina Awais, Zahid Mushtaq and
Abstract
Hemorrhage is spontaneous bleeding caused by a defect in clotting mechanism which fatal to human health. Proteases have important role in human health are helpful in metabolism and in cure of diseases like hemorrhagic disorders. Proteases have a role in heamostasis mechanism in clot formation by the regulation and production of thrombin like enzymes. These enzymes obtained from plant sources which has taken a primary importance in the medical sciences for the treatment of hemorrhagic disorders. During the last few years studies are focused on thrombin like enzymes for isolation, identification, cloning, purification, characterization and overproduction for commercialization in fields like medical sciences, detergent development and meat tenderizer. In our present research an act was made to isolate the gene of cysteine protease a thrombin like enzyme called papain like cysteine proteases (PLCPs) from the leaves of Carica Papaya grown locally. This enzyme has the ability to stop strong procoagulant action and has also been reported to be fibrinogen activator. PCR amplification of PLCPs gene sequence was done by using specifically designed primers (originally for cDNA) using genomic DNA as template that is isolated from the leaves of plant using CTAB method. The amplified product of ~1200 base pairs different from the expected size of 1023 bp was confirmed electrophoretically. The PCR optimized conditions were at 2.5 mM MgCl2 and with initial denaturation at 95°C for 4 minutes and followed by 35 cycles of denaturation at 94°C for 1 minute, annealing 49°C for 1 minute, extension 72°C for 2 minutes and final extension at 72°C for 10 minnutes. The PCR amplicon from genomic DNA of Carica Papaya is not reported previously. This study can further help to understand the genetic diversity of this enzyme, their characteristics, regulation and better research based understanding of the topic.