GST/ AEP 融合蛋白原核表达载体的构建、表达及鉴定

目的：为进一步研究抗癫痫肽（And—epilepsy peptide，AEP）的抗痫机制及筛选其相关作用蛋白，进行GST／AEP融合蛋白原核表达载体的构建及融合蛋白的表达。方法：通过PCR基因扩增对AEP基因进行扩增，并将其克隆于谷胱甘肽-S-转移酶（GST）融合蛋白表达质粒pGEX-4T-1中，经酶切、序列鉴定分析后，用该重组质粒转化大肠杆菌B121（DE3），经IPTG诱导获得表达，并采用Western Blot进行检测。结果：成功构建了AEP原核表达载体，并在大肠杆菌B121中获得表达。结论：成功构建了GST／AEP原核表达载体，并表达了GST／AEP融合蛋白。

Construction and expression of prokaryotic expression vector of GST/ AEP fusion protein and identification

Objective: To express fusion protein of GST and Antiepilepsy peptide(AEP) in E.coli.Methods: The core fragment of AEP was cloned into pGEX-4T-1 cotaining glutathione s-transterase(GST) fusion protein gene.Following the restriction enzyme digestion analysis and sequencing,pGEX-4T-1/AEP was transformed into E.coli Bl21(DE3).GST/AEP fusion protein was expressed under IPTG induction and the AEP protein was identified by the Western-blot.Results: The restriction endonuclease digestion analysis of recombinant plasmid demonstrated that the AEP gene had been exactly inserted in pGEX-4T-1,SDS-PAGE analysis showed that the relative molecular mass of the fusion protein was about 34KD.The GST/AEP fusion protein was expressed in E.coli Bl21(DE3)and identified by the Western-blot.Conclusion: pGEX-4T1/AEP vector was successfully constructed,and the GST/AEP fusion protein in E.coli Bl21(DE3)was expressed.