Cloning and expression of human lysozyme gene in Pichia pastoris

AIM To construct a recombinant plasmid between vector pPIC9K and human lysozyme gene. Active human lysozyme was expressed. METHODS Recombinant plasmid pUC19 hLY was amplified by PCR. It was cloned into expression plasmid pPICPK and it's lined fragment was transformed into electroporated Pichia pastoris strains. Recombinant of human lysozyme and Pichia pastoris was obtained. Was used G418 to screen positive clone, and PCK to identify integration of gene of human lysozyme into genome of Pichia pastoris. The positive clone was expressed in Pichia pastoris strains. RESULTS Sequence of human lysozyme we got different from published sequence. In oredr to get the correct sequence, we divise two primers to correct the mutant gene. The sequence was determined again. This time the result showed that the sequence was 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 induced by Methylotrophic. An anticipated 15 000 protein band appeared on SDS PAGE gel and Western blot and amounted to 0.47 of the total screened protein. The expressed raw product exhibited the antigenicity and activity for human lysozyme. CONCLUSION Recombinant plasmid pPIC9K hLY has been successfully constructed and expressed in Pichia pastoris .