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目的优化家蝇抗真菌肽(MAF-1)原核表达条件及重组蛋白的活性验证。方法利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Quantity One凝胶电泳图像分析系统研究不同的诱导温度、异丙基硫代-β-D半乳糖苷(IPTG)诱导浓度和诱导时间对融合蛋白表达的影响;融合蛋白用镍离子金属螯合剂亲和层析柱进行蛋白纯化,切除His标签后进行活性验证。结果在加入浓度为25μmol/L的IPTG,34℃诱导18 h,融合蛋白的表达量最高;融合蛋白纯化后浓度为0.706 mg/m L,其最低抑杀白色念珠菌的浓度为70μg/m L。结论成功获得重组融合蛋白的最佳表达条件,并纯化目的蛋白,切除His标签后的目的蛋白具有抗真菌活性。
Objective To optimize the prokaryotic expression conditions and the activity of recombinant protein of housefly antifungal peptide (MAF-1). Methods Different temperatures were induced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Quantity One gel electrophoresis (IPE). Isopropylthio-β-D-galactoside The effect of induction concentration and induction time on the expression of fusion protein was studied. The fusion protein was purified by affinity chromatography with nickel ion metal chelate affinity chromatography column, and the His tag was excised for activity verification. Results The fusion protein was expressed at the highest concentration of IPTG at 25μmol / L for 18 h after induction at 34 ℃. The concentration of the fusion protein was 0.706 mg / m L and the minimum inhibitory concentration of Candida albicans was 70 μg / mL . Conclusion The optimal expression conditions of the recombinant fusion protein were successfully obtained. The target protein was purified and the target protein after His tag was excised was antifungal activity.