镇痛活性肽VRD突变体(S54A)的构建与表达以及对活性的影响

目的研究54位丝氨酸对镇痛活性肽(analgesic peptide,AGP)VRD生物活性的影响。方法利用一步聚合酶链式反应(polymerase chain reaction,PCR),用Ala替代镇痛活性肽VRD第54位的Ser,从而构建突变体S54A。采用本实验室构建的pSYPU作为表达载体在大肠杆菌BL21(DE3)中表达重组蛋白。通过金属离子螯合亲和柱色谱和阳离子交换柱色谱方法对重组蛋白进行分离纯化。小鼠扭体法测定重组蛋白的镇痛活性。结果重组蛋白主要以可溶性形式存在,通过两步色谱方法获得了电泳纯样品。突变体S54A的镇痛活性明显低于AGP-VRD(P<0.05)。结论镇痛活性肽VRD中54位丝氨酸与其镇痛活性密切相关。

Construction, expression and the impact of active peptide VRD mutant (S54A) to the analgesic activity

Objective To research the impact of Ser54 in AGP-VRD to the analgesic activity.Methods Using one-step polymerase chain reaction(PCR),the serine at the position 54 was replaced by alanine to construct the mutant AGP-VRDS54A.The AGP-VRD and mutant gene were finally cloned into T7 promoter-based E.coli expression vectors,pSYPU.The recombinant proteins were purified by metal chelating affinity chromatography and cation exchange chromatography,and the analgesic activities of them were detected by mouse-twisting action test.Results The recombinant proteins were expressed in BL21(DE3)in soluble form.The proteins were purified to homogeneity through a two-step chromatography.The analgesic activity of mutant AGP-VRDS54A was significantly lower than that of AGP-VRD(P<0.05).Conclusions The Ser54 is closely related to the analgesic activity of AGP-VRD.