蝎镇痛活性肽AS的一个二硫键对镇痛活性的影响

Impact of a disulfide bridge in scorpion active peptide AS to the analgesic activity

目的研究蝎镇痛活性肽AS中二硫键C12-C62对其生物活性的影响。方法利用一步聚合酶链式反应(polymerase chain reaction,PCR)分别用Ser替代蝎镇痛活性肽AS中第12位、第62位及第12位和第62位的Cys,从而构建其3个突变体。采用金属离子螯合亲和层析方法对表达突变体蛋白进行纯化。采用小鼠扭体法测定3个突变体的镇痛活性。结果构建了3个突变体质粒,在BL21(DE3)中实现了可溶性表达。采用金属离子螯合层析方法获得了电泳纯样品。其镇痛活性明显低于Bmk AS(P<0.01)。结论3个突变体与rBmK AS镇痛活性有明显差异,二硫键C12-C62与蝎毒镇痛活性密切相关。

Objective To research the impact of a disulfide bridge in recombinant scorpion active peptide rBmK AS to the analgesic activity. Methods The cysteines at the 12,62,12 and 62 were replaced by serine to construct three mutants through mutagenesis. The three mutants were purified by metal chelating affinity chromatography. And the analgesic activity of them was detected by an animal model of twisting action of the mice. Result The three mutant genes were constructed. They were cloned and expressed in B121（DE3）. The proteins were soluble. The proteins were purified by means of metal chelating chromatography. The mutant＇s analygesic activity was lower than Bmk AS significantly（ P 〈 0.01）. Conclusions The analgesic activity of those mutants is different with rBmK AS obviously. The disulfide bridge C12-C62 is interrelated closely to the analgesic activity.