菌丝霉素NZ2114基因在毕赤酵母中表达及中试发酵研究

Expression and pilot scale fermentation of plectasin NZ2114 gene in Pichia pastoris

构建表达菌丝霉素NZ2114蛋白的真核重组表达质粒p Pic Zα-NZ2114,并转化毕赤酵母菌GS115,Zeocin抗性筛选阳性重组子,通过PCR鉴定、Tricine-SDS-PAGE分析和琼脂孔穴扩散法筛选获得菌丝霉素NZ2114组成型表达菌株,并采用p H-溶氧监控策略进行工程菌毕赤酵母菌GS-115/p Pic Zα-NZ2114的50 L中试发酵研究。结果表明,本实验成功实现了菌丝霉素NZ2114基因在毕赤酵母GS115中的组成型表达p Pic Zα-NZ2114,Tricine-SDS-PAGE分析显示,在4.4 k Da处有明显的目的条带,工程菌经50 L发酵罐诱导培养96 h后取上清,对金黄色葡萄球菌CMCC26003有明显的抑制作用。菌丝霉素NZ2114基因在毕赤酵母中表达的方法在本研究中成功应用,中试研究为以后的规模化生产和应用奠定了基础。

DNA fragment coded plectasin NZ2114 protein was cloned into p Pic Zα-NZ2114 and the re-combinant plasmid was transformated into Pichia pastoris GS115. The transformants were screened byZeocin and PCR amplification of yeast colonies. The expression supernatant was analyzed by Tricine-SDS-PAGE. The antibacterial activity of expression product was analyzed by agarose diffusion assay.Datas showed that the genetic engineering Pichia pastoris GS115/p Pic Zα- NZ2114, constitutive ex-pressed the recombinant plectasin NZ2114, was successful constructed. The result of Tricine-SDS-PAGE analysis of expression supernatants showed that the line with molecular weight of 4.4 k Da wascorresponding to NZ2114. After cultured for 96 h in 50 L fermenter, the expression supernatant of thePichia pastoris GS115/p Pic Zα-NZ2114 had obvious antibacterial activity on S. aureus CMCC26003,exceed 0.012 3~0.016 3 μg/ml ampicillin. Constitutive expression and pilot scale of plectasin NZ2114 was applied successfully in this study, which laid the fundation for the future large-scale production.