关键词: 毕赤酵母, 基因表达, 人防御素HNP4

Abstract: In order to construct a recombinant plasmid pPIC9K-HNP4 between vector pPIC9K and human defensin HNP4 gene and express active human defensin HNP4, HNP4 gene was amplified with PCR from plasmid pUC18-HNP4, which contains HNP4 sequence and is inserted into the yeast expression vector pPIC9K. The recombinant plasmid was lined fragment by SacI and transformed into Pichia pastoris strains GS115 yeast using electroporation. Transformant of human defensin HNP4 and Pichia pastoris was obtained using G418 to screen positive clone and PCR to identify integration of gene of HNP4 into genome of Pichia pastoris. The yeast transformant was induced by methanol to express human defensin HNP4. The results show that gene of human defensin HNP4 has been integrated into genome of Pichia pastoris with restriction analysis, and PCR identified. The expression of human defensin HNP4 was induced by methanol. An expected 4.0 kDa protein band appeared on SDS-PAGE gel and Western-blotting. The expression level of HNP4 protein in this strain was about 500 mg/L. The expressed raw product exhibited antimicrobial activity. This reveals that recombinant plasmid pPIC9K-HNP4 has been successfully constructed and expressed stably and efficiently in Pichia pastoris.

Key words: gene expression, Pichia pastoris, human defensin HNP4