The Study Of Detection And Pharmacological Activity Of CTB-LK Expressed In Yeast Pichia Pastoris And Sunflower

The thrombus disease especialy in cardiovascular and cerebrovascular is the key reason to the people's die of illness in china. Lumbrokinase(LK) is an important fibrinolytic enzyme derived from earthworm and is effective in treating diseases caused by thrombus. However the low-level utilization of oral dosing prohibits the widespread use of the LK. The nontoxic B-chain of cholera toxin (CTB) subunit is known for its adjuvant properties, when conjugated to various antigens, The adjuvant effect of CTB is dependent on the binding of the pentameric form to the GM1 gangliosides present on cellular surfaces.In our study, we explore if the CTB can significantly increases the ability of LK to treat the thrombus after oral administration due to its specific character.Here, the Pichia pastoris(P. pastoris) expression vector pPIC9k-CTB-LK was constructed, and inclusion of the recognition sequence for the ubiquitous protease furin between CTB and LK allowed for intracellular cleavage of the fusion product and subsequent transport of LK, but not CTB, via the mucosal vasculature.The linker (GGGGS)3 were added between the CTB and LK domains to direct the protein folding. After purification, the CTB-LK which was well expressed by P. pastoris, was found the good fibrinolytic activity and the effective ability to treat the thrombus through the experiment of fibrin-protein plate and the mouse model of thrombus. In addition, we showed that P.pastoris be bioengineered to secrete CTB-LK, which in turn conveniently mediate the encapsulation of yeast cells with silica under ambient temperature and pH.Furthermore, sunflower was transformed by C58 which contained the vector pX6-CTB-LK through agrobacterium-mediated transformation, and 2 seeds were harvested finally.The genomic DNA and RNA was extracted from the leaves of sunflower for PCR and RT-PCR analysis. The results confirmed that the CTB-LK gene had integrated into the genome of the transgenic sunflower plants.In brief, this study establised a good basis for the thrombus disease therapy following oral medication, and it provided a good experiment and theoretical basis for that CTB can significantly increases the ability of LK to treat the thrombus after oral administration .