摘要
目的观察不同信号肽对重组胰高血糖素样肽-1(GLP-1)分泌的影响,筛选高效表达GLP-1的信号肽。方法构建GFP-GCG-GLP-1、GFP-NT4-GLP-1、GFP-EX4-GLP-1重组质粒,根据信号肽不同分为阴性对照(NC)、空载、EX4、NT4和GCG组。使用Vigofect试剂盒转染HEK293T细胞;RT-PCR分析转染后HEK293T细胞GLP-1 mRNA表达水平;ELISA法测定转染后培养基上清液中GLP-1水平。结果转染24 h后HEK293T细胞,检测EX4、NT4、GCG组GLP-1mRNA水平差异无统计学意义,与NT4、GCG组比较,培养基上清液中EX4组GLP-1蛋白浓度升高,差异有统计学意义(P<0.01)。结论 3种信号肽均能引导GLP-1分泌表达,EX-4信号肽引导效率最佳。
Objective To explore the effects of three types of signal peptides on recombinant GLP-1 secretion in development of screening optimal expression vector of GLP-1. Methods The recombinant expression vectors of GFP-GCG-GLP-1, GFP-NT4-GLP-1, GFP-EX4-GLP-1 were constructed and transfected in HEK293T cell with Vigofect. The mRNA expression of GLP-1 was analysed by RT-PCR in HEK293T cell and the protein secretion of GLP-1 was tested by ELISA in the cells supernatant. Results RT-PCR results show that there were no significant differences in the expression levels of GLP-1 among the constructs at 24 h after transfection. The GLP-1 protein se- cretion of EX4 group was significantly higher than other group at 24 h after transfection （P 〈 0. 01 ）. Conclusion The EX4 signal sequence could be effective on promoting the secretion of other heterologous proteins in HEK293T cells.
出处
《安徽医科大学学报》
CAS
北大核心
2013年第7期764-768,共5页
Acta Universitatis Medicinalis Anhui
