人α-防御素5在毕赤酵母中分泌表达的研究

目的 探索人α-防御素5成熟肽(mHD5)在酵母中高效分泌表达的可行性.方法 PCR扩增获得酵母菌偏好的mHD5密码子序列,应用基因重组方法构建表达质粒,电击转化酵母菌株GS115,应用G418浓度梯度、表型和PCR技术筛选高拷贝转化株.经摇瓶发酵和甲醇诱导,Tricine-SDS-PAGE分析重组mHD5(rmHD5)表达量,并采用Western blot鉴定.选用琼脂扩散法检测rmHD5的抑菌活性.结果 构建了pPIC9K-mHD5酵母表达质粒.筛选得到3株His /Mut 表型,且耐受8 mg/ml G418的多拷贝转化酵母菌株,成功获得表达量约120 mg/L的发酵上清.rmHD3对大肠杆菌(EC25922)和金黄色葡萄球菌(SA25923)2种标准菌株均具有明显的抑菌活性.结论 防御素基因可以在毕赤酵母中实现分泌型离表达,此策略可能是抗菌肽工业化开发的有效途径之一.

Secretion expression of human alpha-defensin 5 in Pichia pastoris

Objective To explore the feasibility of high-level secretion expression of bioactive mature human alpha-defensin 5 (mHD5) in Pichia pastoris Methods DNA fragment containing mHD5 coding sequence with biased codons of Pichia pastoris was amplified by PCR,then inserted into the Pichia pastoris expression vector pPIC9K After the recombinant plasmid was transformed into the yeast host strain GS115 by electroporation,the transformants with multi-copy inserts were selected by PCR identification and G418 phenotype screen After optimization of the flask-shaking culture fermentation,the expression of recombinant mHD5 (rmHD5) induced by methanol was analyzed by Tricine-SDS-PAGE and Western blot The bioactivity of rmHD5 was examined by antibacterial assay Results The plasmid pPIC9K-mHD5 was correctly constructed Three transferred GS115 strains containing multi-copy mHD5 gene were singled out,which could grow in the media with 8 mg/ml G418 and showed His /Mut phenotype The yield of rmHD5 was about 120 mg/L in the clarified broth of fermentation media The rmHD5 displayed obvious antibacterial activity against both EC25922 and SA25923 Conclusion The strategy of secretion expression of defensin gene in Pichia pastoris may be an effective way to produce bioactive antimicrobial peptide on a large scale