|本期目录/Table of Contents|

[1]林松峰,张 瑞,王丹丹,等.体外表达GLP-1多拷贝的最优条件研究[J].天津医科大学学报,2015,21(05):388-392.
　LIN Song -feng,ZHANG Rui,WANG Dan-dan,et al.?Optimization for the expression of multi-copy peptides GLP-1 in vitro[J].Journal of Tianjin Medical University,2015,21(05):388-392.
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体外表达GLP-1多拷贝的最优条件研究(PDF)
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
21卷
期数:
2015年05期
页码:
388-392
栏目:
基础医学
出版日期:
2015-09-20

文章信息/Info

Title:
?Optimization for the expression of multi-copy peptides GLP-1 in vitro
文章编号:
1006-8147（2015）05-0388-05
作者:
林松峰1; 3 ; 张 瑞1; 王丹丹2 ; 郭 刚1
(1.天津医科大学代谢病医院内分泌研究所,天津 300070;2. 协和干细胞基因工程有限公司,天津300384；3.天津长和生物技术有限公司，天津 301925）
Author(s):
LIN Song -feng 1; 3 ; ZHANG Rui 1 ; WANG Dan-dan 2 ; GUO Gang1
(1. Institute of Endocrinology Metabolic Diseases Hospital, Tianjin Medical University ,Tianjin 300070, China; 2. Xie He Stem Cell Gene Engineering Company ,Tianjin 300384, China；3.Ever Union Biotechnology Company Limited，Tianjin 301925,China)
关键词:
GLP-1; 多聚体; 基因表达
Keywords:
optimization;  GLP-1;  expression
分类号:
Q7
DOI:
-
文献标志码:
A
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pET-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。
Abstract:
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.

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备注/Memo

备注/Memo:
作者简介 林松峰（1983-），男，技术员，硕士在读，研究方向：生物化学与分子生物学 ；
通信作者：郭刚 E-mail：guogangtj@126.com。
更新日期/Last Update: 2015-09-21