枯草芽孢杆菌B111中性蛋白酶基因BS2在毕赤酵母中的表达

Cloning and Expression of a Neutral Protein Gene BS2 from Bacillus subtilis B111 in Pichia pastoris

【目的】为开发抗病基因和生物农药新资源,纯化鉴定枯草芽孢杆菌B111分泌的抗棉花黄萎病菌蛋白质,克隆其编码基因,并分析在毕赤酵母中的表达特性。【方法】通过超滤浓缩、90%硫酸铵沉淀、葡聚糖凝胶QAE-A50强阴离子交换柱洗脱及冻干等步骤,分离纯化抗菌蛋白。采用联机反相毛细管柱液相色谱电喷雾串联质谱,鉴定抗菌蛋白种类。克隆包含前导区序列在内的抗菌蛋白基因序列,构建表达载体pPIC9K-PT,转化毕赤酵母GS115。转化子经PCR鉴定,甲醇诱导表达产物经中性蛋白酶活性和抗菌活性检测验证功能。【结果】纯化鉴定了一个来源于枯草芽孢杆菌B111的抗菌中性蛋白酶BS2。克隆了包含前导区序列在内的抗菌蛋白基因BS2,并在毕赤酵母中获得有效表达。重组BS2蛋白分子量约69kD,表达水平29.77mg·L-1,水解酪素酶活力27800U·g-1,并显示抗黄萎病菌活性。【结论】纯化鉴定了一个具有抗菌活性的枯草芽孢杆菌中性蛋白酶BS2,克隆其编码基因BS2,并成功实现在酵母中的有效表达。

[Objective] In order to exploit novel disease-resistance gene and biological pesticide sources, antifungal protein secreted by Bacillus subtilis B 111 was purified and identified, then its gene was cloned and expressed in Pichia pastoris. [ Method] Antifungal protein was purified by ultrafiltration, 90% ammonium sulfate precipitation and QAE Sephadex A50 ion exchange chromatogram column, then it was identified via CapLC-ESI-MS-MS. Its gene encoding antifungal protein was cloned. The recombinant expression vector pPIC9K-PT was constructed and transformed into P. astoris GSll5. The transformants were identified by PCR analysis. The products of the transformants induced by methanol were identified by neutral protease and antifungal activity assay. [ Result] An antifungal protein BS2 was purified and identified to be a neutral protease from B. subtilis. Antifungal gene BS2 with pro-sequence was cloned. The recombinant expression vector pPIC9K-PT was constructed and BS2 gene was expressed in P. pastoris. The recombinant BS2 protein exhibited a molecular mass of approximately 69 kD and presented neutral protease activity of 27 800 U.g^-1 and antifungal activity. Its production reached up to as high as 29.77 mg·L^-1. [Conclusion] In this study, an antifimgal neutral protein BS2 was purified and identified, then its gene was cloned and successfully expressed in P. pastoris.