体外表达GLP-1多拷贝的最优条件研究  

Optimization for the expression of multi-copy peptides GLP-1 in vitro

作　　者：林松峰[1,2] 张瑞[1] 王丹丹 郭刚[1] 

机构地区：[1]天津医科大学代谢病医院内分泌研究所,天津300070 [2]天津长和生物技术有限公司,天津301925 [3]协和干细胞基因工程有限公司,天津300384

出　　处：《天津医科大学学报》2015年第5期388-392,共5页Journal of Tianjin Medical University

摘　　要：目的:探讨胰高血糖素样肽-1(GLP-1)的序列拷贝数及拷贝温度、时间与其体外表达水平的关系。方法:首先将以4拷贝数为单位的GLP-1序列插入到p ET-22b(+)中构建GLP-1多拷贝表达载体,然后将表达载体转化至大肠杆菌BL21(DE3)中进行蛋白体外培养表达。取不同温度和诱导时间段进行蛋白表达,检测GLP-1在不同条件下的蛋白表达量,优化蛋白最适表达条件。结果:GLP-1多拷贝在BL21菌株中的最佳表达温度为26℃,最适诱导时间为8 h。拷贝数与GLP多拷贝表达量呈负相关性,拷贝数越多,蛋白产量越低。结论:GLP多拷贝最佳体外表达条件为4拷贝,26℃培养IPTG诱导8 h。Objective：To investigate the relationship between production and copy number coding sequence of the glucagon-like peptide-1 （GLP-1）. Methods：Firstly, four repeated sequences for multi-copy GLP-1 peptides were linked as a motif and inserted into the vector pET-22b （＋）. The completed plasmid including different numbers of GLP-1 copy was subsequently transformed into E.coli BL21 cells, which can be induced to express the interesting peptides by the reagent IPTG （Isopropyl-beta-D-thiogalactopyranoside）. The expression of multi-copy GLP-1 was employed to optimize the fermentation. The conditions such as temperature and inducing time for highest protein yield were analyzed. Finally, the effects of different GLP-1 motif were confirmed to improve the production. Results：Multi-copy GLP-1 was expressed and secreted by E.coli in different conditions, the result demonstrated that a climax yield for protein was obtained at 26℃and 8 hours. Moreover, the production was negatively correlated with copy number of GLP-1. With the multi-copy, the production of peptides marked a significant decline. Conclusion：The final optimized condition to express multi-copy GLP-1 peptides is E.coli BL21 containing 4 copy motif vector cultured at 26℃with inducing time of 8 hours.

关 键 词：GLP-1 多聚体 基因表达 

分 类 号：Q7[生物学—分子生物学]