Cloning Of Human Lysozyme Gene And Expression In Pichia Psatoris

Lysozyme(LY),virtually ubiquitous throughout nature, is considered to be a constituent of unspecific defense mechanism associated with the monocyte-macrophage system. It catalyzes the hydrolysis of the (3 -1,4 glycosidic bond of complex polysaccharides which are the principal constituents of the cell wall of bacteria. Lysozyme has long been used as a model system for protein structure, enzyme kinetic,molecular immunology and molecular evolution studies. Human lysozyme (hLY) is a kind of lysozymes, it participate in the body defense mechanism.lt acts as a shield against bacterial infection and it has effects on malignant growth.Conventionally hLY is obtained from human milk or placenta by small-scale.Because of the limitation of its source,wecan not get it by large-scale.Now by DNA recombinant method,we can get large-scale of human lysozyme from bacteria or yeast.The methylotrophic yeast Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. It has a lot of advantages as expression hosts for the existence of well-established fermentation methods and the presence of methnol-regulated promoters. It not only has wide commercial application, but only has potential signficance in theory.To construct a recombinant plasmid between vector pPIC9K and human lysozyme gene. Activity human lysozyme will be expressed. The recombinant plasmid pUC19-hLY was amplified by PCR. It was cloned into expression plasmid pPICPK and it's lined fragment transformed into electroporated Pichia pastoris strains.Recombinant of human lysozyme and Pichia pastoris was obtained. To use G418 screened positive clone, whether gene of human lysozyme integrate into genome of Pichia pastoris with PCR identified. The positive clone expressed in Pichia pastoris strains. The sequence of human lysozyme we got different from published sequence.In order to get the correct sequence,we divise two primers to correct the mutant gene.The sequence was determined again.This time the result shows that the sequence is in accordance with the published sequence .Gene of human lysozyme have been integrated into genome of Pichia pastoris with PCR identified.The expression of human lysozyme was-6-induced by Methylotrophic.A anticipated 15 000 protein band appeared on SDS-PAGE gel and Western blot and amounted to 47.1% of the total secreted protein.The expressed raw product exhibited the antigenicity and activity for human lysozyme. The recombinant plasmid pPIC9K-hLY has successfully constructed and expressed in Pichia pastoris.