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目的研究蝎镇痛抗肿瘤缬精甘肽(analgesic-antitumor peptide,AGAP)中2个氨基酸残基对其镇痛活性的影响。方法利用已构建成功的2个突变体W38G和R58D,采用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进行纯化。采用小鼠醋酸扭体法测定两个突变体的镇痛活性。结果在大肠杆菌BL21(DE3)中实现了可溶性表达。采用金属离子螯合层析和阳离子交换层析方法获得了电泳纯样品。突变体W38G镇痛活性与未突变重组蛋白(recombinant analgesic-antitumor peptide from Buthus martensii Karschr,BmK AGAP)相同,突变体R58D镇痛活性降低。结论突变体与rBmK AGAP相比镇痛活性发生变化,说明所选取的突变位点对蝎镇痛抗肿瘤缬精甘肽的镇痛活性具有一定的调节作用。
Objective To study the effect of two amino acid residues in analgesic-antitumor peptide (AGAP) on the analgesic activity of scorpion analgesia. Methods Two mutant W38G and R58D were successfully constructed, and the mutant protein was purified by metal ion chelate affinity chromatography and cation exchange chromatography. The analgesic activity of the two mutants was determined using mouse acetic acid writhing assay. As a result, soluble expression was achieved in E. coli BL21 (DE3). Electrophoretic samples were obtained by metal ion chelation chromatography and cation exchange chromatography. The analgesic activity of the mutant W38G was the same as that of the recombinant analgesic-antitumor peptide from Buthus martensii Karschr (BmK AGAP), and the analgesic activity of the mutant R58D was decreased. Conclusion The mutants showed a change in the analgesic activity compared with that of rBmK AGAP, indicating that the selected mutants have some regulatory effects on the antinociceptive activity of the antineoplastic valproic peptide.