牛气管抗菌肽在毕赤酵母中的表达与鉴定

Expression and identification of bovine tracheal antimicrobial peptide in Pichia pastoris

为了在毕赤酵母中获得牛气管抗菌肽(bTAP)的表达,根据已发表的牛气管抗菌肽序列和毕赤酵母对密码子的偏爱性设计2对引物,采用重叠延伸PCR法获得bTAP DNA序列,将其与毕赤酵母(Pichia pastoris)pPIC9K质粒连接,构建表达载体pPIC9K-bTAP。用SalⅠ酶切线性化pPIC9K-bTAP质粒后电转化毕赤酵母GS115,经G418筛选阳性克隆,并用甲醇诱导其表达。SDS-PAGE分析结果表明表达的重组蛋白质分子量正确,抑菌试验结果表明表达的重组蛋白质bTAP对金黄色葡萄球菌具有良好的抑菌效果。

To express bovine tracheal antimicrobial peptide（bTAP） in methylotrophic yeast Pichia.pastoris,two pairs of primers were designed according to the published sequences for bTAP and the codon bias of P.pastoris.The bTAP gene was amplified by overlapping PCR and cloned into the vector pPIC9K to construct the yeast secretory expression vector pPIC9K-bTAP.The recombinant plasmid digested by restriction enzyme SalⅠ was transformed to P.pastoris GS115,and positive clones were screened with G418 resistance and was induced by 0.5% methanol for expression.SDS-PAGE confirmed the correct molecular weight of recombinant expression protein.Antibacterial assay showed that bTAP had strong antimicrobial activity against Staphylococcus aureus.