芋螺毒素MⅦA与GST的融合表达及其镇痛活性的测定  

Fusion expression of conotoxin MⅦA and GST and its analgesic activity

作　　者：陈永对[1] 詹金彪[2] 

机构地区：[1]绍兴文理学院医学院生化教研室,浙江绍兴312000 [2]浙江大学医学院生化教研室,浙江杭州310058

出　　处：《中国现代医学杂志》2008年第6期729-732,共4页China Journal of Modern Medicine

基　　金：浙江省科技厅重点项目011103008;浙江省卫生厅项目2000G002;杭州市科委项目2001123B31

摘　　要：目的构建芋螺毒素MⅦA基因的融合表达质粒pGEX-2T/CTXMⅦA在大肠杆菌中表达,并对融合蛋白的镇痛活性进行测定。方法根据芋螺毒素MⅦA的氨基酸序列,按大肠杆菌偏爱密码子化学合成CTX MⅦA基因。把合成的CTX MⅦA基因克隆到谷胱甘肽S-转移酶(GST)融合蛋白表达载体pGEX-2T中,构建质粒pGEX-2T/CTX MⅦA。将此质粒转化大肠杆菌BL21,经IPTG诱导后获得融合蛋白GST-CTX MⅦA,用谷胱甘肽偶联的Sepharose 4B柱对融合蛋白进行纯化。热板法测定其对小鼠的镇痛活性。结果获得的融合蛋白GST-CTX MⅦA使小鼠疼痛阈值有显著提高。结论基因工程产生的GST-CTX MⅦA融合蛋白具有明显的镇痛活性,且镇痛活性具有浓度依赖性,可以为进一步的科研及临床应用开辟新的途径。[Objective] To construct fusion expression plasmid pGEX-2T/MⅦA, then transform it into E. coli BL21 for expression, and evaluate the analgesic effect of the fusion protein on mice. [Methods] E. coil-preferable codon was employed to design the artificial gene of w-conotoxin MⅦA according to the amino-acid sequence. The DNA sequence encoding w-conotoxin M Ⅶ A was synthesized and subsequently cloned into the expression vector pGEX-2T. The fusion protein was expressed in E. coli BL21 and affinitively purified on a Glutathione-Sephamse 4B column. The analgesic activity of fusion protein in mice was determined by the classic hot-plat method. [Results] The fusion protein was obtained and the pain thresholds were markedly elevated when the fusion toxin was intracranially administered to mice. [Conclusions] The fusion protein produced by genetic engineering shows strong dose-dependent analgesic activity. The fusion toxin can be easily produced in E. coli for research and clinical application.

关 键 词：芋螺毒素 镇痛 钙离子通道 GST 

分 类 号：R318[医药卫生—生物医学工程]