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目的研究二硫键C16-C36和C22-C46对镇痛抗肿瘤缬精甘肽BmK AGAP镇痛活性的影响。方法使用本实验室已构建成功的2个二硫键双突变体(C16S,C36S)和(C22S,C46S),在大肠杆菌中诱导表达,通过金属离子螯合亲和层析和阳离子交换层析方法对突变体蛋白分离纯化,采用小鼠醋酸扭体法测定2个突变体的镇痛活性,圆二色谱分析BmK AGAP和2个二硫键突变体的二级结构变化差异,利用生物信息学的方法模建BmK AGAP的三维空间结构。结果 a.目的蛋白在大肠杆菌BL21(DE3)中实现了可溶性表达;b.采用金属离子螯合亲和层析和阳离子交换层析方法获得了电泳纯样品;c.2个双突变体(C16S,C36S)和(C22S,C46S)的镇痛活性与阳性对照组rBmKAGAP比较均下调,具有显著性差异。结论二硫键C16-C36和C22-C46在保持和调节BmK AGAP的镇痛生物活性方面具有重要作用。
Objective To study the effect of disulfide bond C16-C36 and C22-C46 on analgesic activity of analgesic anti-tumor valomide peptide BmK AGAP. METHODS: Two disulfide double mutants (C16S, C36S) and (C22S, C46S) that had been successfully constructed in our laboratory were used to induce the expression in E. coli. The metal ions chelated affinity chromatography and cation exchange chromatography Methods The mutants were isolated and purified. The analgesic activities of the two mutants were determined by mouse acetic acid writhing assay. The secondary structure changes of BmK AGAP and two disulfide mutants were analyzed by circular dichroism. Bioinformatics Approach to modeling the 3D spatial structure of BmK AGAP. Results a. The target protein was expressed in Escherichia coli BL21 (DE3) in a soluble form; b. Metal ion chelated affinity chromatography and cation exchange chromatography were used to obtain the pure electrophoresis samples; c.2 double mutants (C16S , C36S) and (C22S, C46S) compared with the positive control group rBmKAGAP were significantly lower, with significant differences. Conclusion The disulfide bonds C16-C36 and C22-C46 play an important role in maintaining and regulating the analgesic bioactivity of BmK AGAP.