泥鳅抗菌肽在毕赤酵母SMD1168中的高效表达及活性检测

High-level expression in Pichia pastoris SMD1168 of misgurin and its antimicrobial activity

摘要:

【背景】泥鳅抗菌肽Misgurin是泥鳅非特异性免疫防御系统的重要组成部分，具有广谱和较强的抗菌能力，所以获得大量抗菌肽是很有必要的。【目的】为了实现高效表达泥鳅抗菌肽Misgurin。【方法】将泥鳅抗菌肽的目的基因与pPIC9K表达载体连接，构建重组表达质粒pPIC9K-misgurin，Sal I酶切线性化，再通过电击法将其整合到毕赤酵母SMD1168染色体上。在MD固体培养基挑选阳性克隆子到MD液体培养基中，30 °C、200 r/min摇瓶培养96 h，转接到BMMY液体培养基进行诱导表达，每隔24 h加入5%的甲醇。通过比较抑制大肠杆菌和金黄色葡萄球菌抑菌圈直径的大小，筛选出活性较高的菌株pPIC9K-misgurin-22。将该菌株在100 L的发酵罐中诱导表达，48 h后进行活性检测。【结果】经Tricine-SDS-PAGE蛋白胶检测和质谱分析鉴定，pPIC9K-misgurin-22菌株诱导表达的活性物质为抗菌肽Misgurin。发酵罐发酵48 h相对于摇瓶发酵48 h，抗大肠杆菌的生物效价提高了1.47倍，抗金黄色葡萄球菌的生物效价提高了1.43倍；抗菌肽Misgurin对鲍曼不动杆菌、沙门氏菌有较弱的抗菌活性，对致病菌产气荚膜梭菌和益生菌如枯草芽孢杆菌、粪肠球菌、乳酸菌没有抗菌活性，无明显溶血活性；当温度达到90 °C时发酵液的抗菌活性明显减弱，调发酵液的pH值在1.0?12.0之间都有抗菌活性；加入胰蛋白酶、胃蛋白酶、蛋白酶K后抗菌活性减弱。【结论】获得了一株在毕赤酵母中表达量较高、有工业化生产潜力的产抗菌肽Misgurin的菌株。

关键词:泥鳅抗菌肽，毕赤酵母，发酵，抑菌活性，溶血活性，耐酸碱

Abstract:

[Background] Misgurin is an important component of nonspecific immune defense system in Misgurnus anguillicaudatus. It has broad spectrum and strong antimicrobial ability. Therefore, it is necessary to obtain large quantities of antimicrobial peptides. [Objective] To achieve high-efficient expression of misgurin. [Methods] Misgurin gene was cloned into expression vector pPIC9K in order to construct recombinant expression plasmid pPIC9K-misgurin. Using Sal I restriction endonuclease digest linearization scheme, the recombinant expression vector was integrated into the chromosome of Pichia pastoris SMD1168 by electric shock method. The positive single colonies was screened through minimal dextrose (MD) solid medium, which was inoculated MD liquid medium at 30 °C, 200 r/min shake flask for 96 h before transfer to BMMY liquid medium to induce expression, adding 5% methanol every 24 h. By comparing the diameter of bacteriostasis circle of Escherichia coli and Staphylococcus aureus, strain pPIC9K-misgurin-22 with the better antimicrobial activity was screened out and inoculated to a 100 L of fermentor induction for 48 h and then detected of activity. [Results] This active substance of pPIC9K-misgurin-22 strain was identified as misgurin by Tricine-SDS-PAGE protein gel detection and mass spectrometry analysis. In comparison with shaking flask fermentation, the bioavailability of Escherichia coli and Staphylococcus aureus in fermentor fermentation increased by 1.47 fold, 1.43 fold, respectively; misgurin has weak antimicrobial activity to Acinetobacter baumannii and Salmonella. It has no antimicrobial activity to Clostridium perfringens and probiotics (Bacillus subtilis, Enterococcus faecalis, Lactobacillus), and has no obvious hemolytic activity; When the temperature reaches 90 °C, the antimicrobial activity of fermentation broth is obviously weakened, the pH value of fermentation broth at 1.0?12.0, everyone has antimicrobial activity; The antimicrobial activity is weakened after adding trypsin, pepsin and protease K. [Conclusion] A strain producing misgurin is obtained, which has high expression and industrialized production potential in Pichia pastoris.

Key words:Misgurin, Pichia pastoris, Fermentation, antimicrobial activity, Hemolytic activity, Tolerance acid and alkali