Exendin-4与绿色荧光蛋白融合基因的表达与活性研究

目的:表达具有双功能特性的Exendin-4-GFP融合蛋白。方法:构建融合表达载体pET21a( )/Exendin-4-GFP,转化大肠杆菌BL21后,以IPTG诱导融合蛋白的表达,并利用镍金属螯合层析树脂对其进行纯化。采用胰高血糖素样肽-1受体(GLP-1R)转染的CHO细胞考察融合蛋白的结合特性,并检测其生物活性。结果:含有融合表达载体的重组菌株诱导表达后,经SDS-PAGE及免疫印迹分析显示,31.0ku的融合蛋白在大肠杆菌中得到了表达,且具有Exendin-4的抗原活性。受体结合实验表明,该融合蛋白能够与GLP-1R特异性结合,在488nm激发光下呈现绿色荧光。体内活性实验表明,融合蛋白具有明显的降血糖活性。结论:本研究表达的Exendin-4-GFP融合蛋白同时具有Exendin-4的活性和荧光特性,为研究GLP-1受体功能以及Exendin-4与细胞相互作用的机制奠定了基础。

Expression and Characterization of Exendin-4 GFP Fusion Protein

Objective: To investigate the expression and function of Exendin-4 green fluorescent proteins(GFP) and fusion protein. Methods: The fusion expression vector pET21a( )/Exendin-4-GFP was constructed and transformed to E.coli BL21. The expressed fusion protein after induction with IPTG was purified by nickel chelation chromatography. The binding ability of fusion protein was determined with CHO cells transfected by GLP-1R,and the bioactivity was detected in rats. Results: SDS-PAGE and Western blot analysis showed that the 31.0 ku fusion protein was expressed correctly in E.coli. The receptor binding experiments indicated that the fusion protein with GFP activity could bind especially to GLP-1R. The fusion protein showed significant glucose-lowering action in rats. Conclusion: The fusion protein prepared in this study has not only the green fluorescence but also the Exendin-4 activity. The results provided reliable basis for the research on functional mechanism of GLP-1 receptor and its reaction with cells.