重组枯草芽孢杆菌素subtilosin A基因的合成及其在毕赤酵母中的表达

Synthesis of recombinant gene for Bacillus subtilis bacteriocin subtilosin A and its expression in Pichia pastoris

人工改造并合成subtilosin A的结构基因(rsbo A),并在毕赤酵母中诱导表达重组蛋白。结果表明:经阳性大肠杆菌转化子菌落PCR和测序鉴定,表明重组质粒p PIC9K-sbo构建正确;经阳性酵母转化子的菌落PCR产物电泳检测,可见清晰目的基因条带,证明重组酵母GS115-p PIC9K-sbo构建正确;经枯草芽孢杆菌素蛋白电泳检测,可见明显蛋白条带,表明重组rsbo A基因在毕赤酵母GS115中获得了分泌表达。在抑菌效果检测中,重组酵母工程菌的发酵液对铜绿假单胞菌具有一定的抑菌活性。本研究开创了有望替代抗生素的小分子肽细菌素异源表达新思路,为枯草芽孢杆菌素subtilosin A的工业化生产奠定了基础。

In this study,the recombinant gene for subtilosin A（rsbo A） was synthesis for the sake of the expression for this bacteriocin in host cell P. pastoris. Both the results of colony PCR of positive Escherichia coli recombinants and DNA sequencing indicated that recombinant plasmid p PIC9K- sbo was constructed successfully. In addition,the results of colony PCR of positive P. pastoris recombinants indicated that recombinant Pichia GS115- p PIC9K- sbo was constructed successfully. Protein electrophoresis analysis of positive P. pastoris recombinants displayed that the recombinant gene rsbo A was secretory expressed in P. pastoris GS115.Analysis of antimicrobial activity showed that the fermentation broth of this recombinant yeast possess antibacterial activities against P. aeruginosa. The result of this work built a new strategy of bacteriocins,a small molecule peptide,in heterologous expression. The study laid a foundation for industrialized production of Bacillus subtilis antibiotic peptide subtilosin A.