Expression Of Fusion Protein CTB-LK In Soybean Bioreactor

Plant derived protein pharmaceuticals offer many advantages, including low cost of production, storage and transportation, heat stability, absence of human pathogens, protection of proteins in the stomach through bioencapsulation, elimination of the need for expensive purification and sterile injections.However, the major limitation in the efficient delivery of plant-expressed therapeutic proteins across the intestianal epithelial layer is their permeability. Recepror-mediated oral delivery might serve as a possible route for delivery of biopharmaceutical proteins.In this work, we utilize cholera toxin B-subunit(CTB) as a transmucosal carrier. Ctb and Lembrokinase(LK) gene, separated by a linker peptide, was expressed in soybean cells. LK is a kind of proteases, extracted from earthworm, commonly used for melting thrombosis in medicine.LK and CTB gene was cloned into plant expression vector pBI121, then the vector was transformed into Agrobacterium tumefaciens C58 with electorphrosis.Agrobacterium-mediated soybean cotyledonary transformation system was refined in several aspects. In order to obtain soybean cotyledonary node explants with high quality, a new method was developed for soybean seeds sterilization, which is different from traditional ways in its solution-free. In this method, soybean seeds was sterilize for 16h using chlorine gas produced by mixing HCl and commercial bleach in a tightly sealed deciccator. Adding thiol compounds in co-culture media enhanced transformation efficiency by inhibiting resistant reaction of plant cells to Agrobacterium. Rooting rate of soybean shoots was improved by reducing IBA content to 0.2mg/L in the rooting medium.After shoot induction, elongation and rooting in media with 150mg/L kanamycin, 22 soybean seedings was transplanted. Through PCR analysis, it was proved that 2 plants were positive.