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目的首次报道利用东亚钳蝎镇痛抗肿瘤缬精甘肽-BmKAGAP产生两种不同的融合蛋白,目的是获得镇痛和抗肿瘤的双功能活性。方法融合蛋白是以嵌合毒素的形式进行构建,由三部分组成,包括促性腺激素释放激素(luteinizing hormone-releasing hormoneL,HRH),两种不同类型的柔性连接物,以及BmKAGAP。克隆编码两种融合蛋白的基因并在大肠杆菌中实现可溶性表达。通过金属离子螯合亲和层析和阳离子交换层析方法对融合蛋白分离纯化,采用小鼠醋酸扭体法和MTT法测定融合蛋白的镇痛活性与细胞抑制活性。结果融合蛋白在小鼠模型中体现出镇痛活性(rLHRH-Tag(His)AGAP>rTag(His)-LHRH-L8-AGAP>rBmKAGAP),同时对肝癌细胞株Hep3B具有细胞毒活性(rLHRH-Tag(His)-AGAP>rBmKAGAP>rTag(His)-LHRH-L8-AGAP)。结论得到优化的双功能融合蛋白候选者为嵌合毒素rLHRH-Tag(His)-AGAP。
OBJECTIVE: It was first reported that two different fusion proteins were generated using the analgesic anti-tumor valproate-BmKAGAP from East Asia, with the aim of obtaining analgesic and anti-tumor bifunctional activities. Methods The fusion protein was constructed as a chimeric toxin and consisted of three parts, including luteinizing hormone-releasing hormone (HRH), two different types of flexible linkers, and BmKAGAP. The genes encoding both fusion proteins were cloned and soluble expression was achieved in E. coli. The fusion protein was isolated and purified by metal ion chelate affinity chromatography and cation exchange chromatography. The analgesic activity and cytostatic activity of the fusion protein were determined by mouse acetic acid writhing assay and MTT assay. RESULTS: The fusion protein showed analgesic activity in mouse model (rLHRH-Tag (His) AGAP> rTag (His) -LHRH-L8-AGAP> rBmKAGAP) and its cytotoxic activity against Hep3B hepatocellular carcinoma cell line (His) -AGAP> rBmKAGAP> rTag (His) -LHRH-L8-AGAP). Conclusion The candidate of optimized bifunctional fusion protein is chimeric toxin rLHRH-Tag (His) -AGAP.