猪源抗菌肽PBD-1在毕赤酵母中的表达及鉴定

Expression and Identification of Porcine Antimicrobial Peptide PBD-1 in Pichiapastoris

本试验旨在实现猪β防御素1(poricine-β-defensin 1,PBD-1)在毕赤酵母中的表达,获得有抗菌活性的抗菌肽PBD-1。根据PBD-1的氨基酸序列和酵母密码子偏好性,设计优化其核苷酸序列,利用SOE-PCR技术获得PBD-1基因序列,克隆到毕赤酵母表达载体pPIC9K中,构建重组质粒pPIC9K-PBD-1,经SacⅠ线性化后转入毕赤酵母SMD1168中。PCR筛选得到阳性酵母表达菌株,经甲醇诱导后得到分子质量约4.5ku的抗菌肽PBD-1。抗菌特性研究结果表明,表达产物抗菌肽PBD-1对大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌均有较好的抑制效果。

In order to produce antimicrobial peptide PBD-1with bioactivity in Pichia pastoris,according to published amino acid sequence of PBD-1and the partiality codon of yeast,the PBD-1gene was amplified by SOE-PCR and cloned into pPIC9 Kto construct a recombinant expression vector pPIC9K-PBD-1.The recombinant vector was linearized by SacⅠ,and then transformed into SMD1168 by electroporation.Positive yeast expression strain was obtained by PCR.The antimicrobial peptide PBD-1（approximately 4.5ku）was expressed by methanol induction.Antibacterial activity assay showed that the antimicrobial peptide PBD-1had better antibacterial activity against E.coli,S.aureus and B.subtilis.