摘要
用大鼠胰高血糖素样肽-1受体(GLP-1R)基因转染带有增强型绿色荧光蛋白(EGFP)报告基因的CHO 细胞,成功地构建了基于 G 蛋白偶联受体信号传导途径的 CHO/EGFP/GLP-1R 检测细胞系,该细胞系能功能性表达 GLP-1R 并且能通过 cAMP 偶联的 EGFP 报告基因的表达评价 GLP-1R 激动剂的活性,利用该细胞系本文进一步对5种 GLP-1与金黄色葡萄球菌蛋白 A(SPA)不同形式连接的融合蛋白进行了体外活性评价,结果表明,5种融合蛋白的活性均低于天然的 GLP-1,将 GLP-1融合在 SPA 的 N 端与 C 端融合相比,能显著的提高其生物活性,在融合蛋白之间加入不同长度的重复序列(GGGGS),有助于提高其活性,但间隔序列的长短对活性没有显著的影响.以上结果表明,利用 CHO/EGFP/GLP-1R 细胞系,能够对 GLP-1及其类似物的体外活性进行定性和定量的分析,为筛选和开发长效 GLP-1类似物奠定了方法学基础。
In this study, rat glucagons-like peptide-1 receptor (GLP-1R) cDNA was transfected into the CHO cells containing enhanced green fluorescent protein (EGFP) reporter gene. The recombinant CHO/EGFP/GLP-1R cell line, which utilized thesignal transduction pathway of G protein-coupled receptor, could functionally express GLP-1R and assay the activity of GLP-1R agonist by cAMP-responsive EGFP gene expression. Five GLP-1 fusion proteins comprising staphylococcal protein A (SPA) by different joint means were evaluated for GLP-1R activation with recombinant cell line, which showed similar activity, but not as effective as native GLP-1. Among the GLP-1 fusions, the C-terminal SPA fusion could Significantly enhance the activity compared to the N-terminal fusion. Besides, fusion proteins with repeated linker sequences (GGGGS) were more effective than those without it, but the length of linker did not significantly embody the activity enhancement. It was concluded that CHO/EGFP/GLP-1R cell line could qualitatively or quantitatively verify the bioactivity of GLP-1 and its analogues. Preliminary studies show a new potential for developing the long-acting GLP-1 analogs for clinical application.
出处
《南开大学学报(自然科学版)》
CAS
CSCD
北大核心
2007年第1期92-98,共7页
Acta Scientiarum Naturalium Universitatis Nankaiensis
基金
应用基础及前沿技术研究计划重点项目