Construction Of Two Expression Vectors Of Gene Mae And Misgurin And Their Expression In Pichia.Pastoris

MAE designed by our lab is a hybrid peptide with ideal antibacterial activity. It derives from MagaininII(amino acid residues 4-14) and Melittin (amino acid residues 2-13), and is optimized according to the relations between the structure and function. In this article, the advanced structure of hybrid peptide MAE is predicted with software. The fused gene mae-intein-cbd is amplified by PCR with the template of plasmid pTYB2, and then it is cloned into expression vector plasmid pPIC9K. After verified by restriction enzyme analyzing and sequencing, the vector is transferred into the eukaryotic host (yeast Pichia. Pastoris strains GS115) with electronic pulse. The recombinant is obtained through culturing on nutrition deficiency medium, and then the strains with multiple copy foreign gene is screened by G418 resistant ability. The strains with high expression level is selected according to the result of expression experiment with the methods introduced by expression guideline, and its expression condition is optimized in three aspects, namely time of inducing, pH value and inducing concentration of methanol. The large-scale expression product is concentrated and desalted by ultra-filter, and the target protein is separated by affinity chromatography with chitin, and then cut by CNBr. The purified product appears evident antibacterial activity with method of agar diffusion.Misgurin is a kind of pepetide separated from loach with high antibacterial activity as well as low hemolytic activity. As a result, it could be developed to be a new antibacterial agent. In this article, the misgurin gene and adaptor are synthesized according to the amino acid sequence reported in the GENEBANK and the need of construction and expression. Adopted a new strategy, multiple copy gene is ligated in the same direction. And then it is cloned into expression vector plasmid pPIC9K. After verified by restriction enzyme analyzing and sequencing, the vector is transfered into the eukaryotic host (yeast Pichia. Pastoris strains GS115) with electronic pulse. The recombinant is obtained through culturing on nutrition deficiency medium, and then the strains with multiple copy foreign gene is screened by G418 resistant ability.The expression experiment is carried out with the methods introduced by expression guideline. According to the result of SDS-PAGE, the target protein is expressed successfully in the Pichia. Pastoris.