人防御素HNP4在毕赤酵母中的表达

Expression of Human Defensin HNP4 in Pichia Pastoris GS115

为了构建人防御素HNP4重组毕赤酵母(Pichia pastoris)表达载体pPIC9K-HNP4,建立高效、稳定表达人防御素HNP4的毕赤酵母表达菌株,并对表达产物进行检测与分析.利用PCR技术从pUC18-HNP4上扩增人防御素HNP4,构建人防御素HNP4重组表达载体pPIC9K-HNP4.SacI线性化后电击法转化入毕赤酵母GS115中,再通过同源重组到酵母染色体上,用G418筛选高表达菌株,在三角摇瓶中0.5%甲醇诱导表达.SDS-PAGE和Western-blotting检测表达产物,用标准大肠杆菌、金黄色葡萄球菌菌株混菌板鉴定活性.结果显示,SDS-PAGE和Western-blotting可检测到大小4.0 kDa左右的目的蛋白条带,表达量可达到500 mg/L,表达的重组人防御素HNP4具有较强的杀菌或抑菌活性.提示成功构建人防御素HNP4重组毕赤酵母表达载体pPIC9K-HNP4,并建立高效、稳定表达有明显抗菌活性的重组人防御素HNP4的毕赤酵母表达菌株.

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.