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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (12): 15-20    DOI: DOI:10.13523/j.cb.20161203
研究报告     
重组人胰高血糖素样肽-1类似物的分离纯化和鉴定
高相雷, 林树珊, 龚庆伟, 潘兰, 马利, 冯艳, 林小鹊, 曾剑, 李文佳, 陈小锋, 陈英
广东东阳光药业有限公司 东莞 523000
Purification and Characterization of Recombinant Human Glucagon-like Peptide-1 Analogue
GAO Xiang-lei, LIN Shu-shan, GONG Qing-wei, PAN Lan, MA Li, FENG Yan, LIN Xiao-que, ZENG Jian, LI Wen-jia, CHEN Xiao-feng, CHEN Ying
Guangdong HEC Pharmaceutical Co., Ltd. Dongguan 523000, China
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摘要：

将合成的人胰高血糖素样肽-1类似物基因插入到原核表达质粒pGEX-4T-3中，构建成rhGLP-1类似物与谷胱甘肽巯基转移酶（glutathione-S-transferases，GST）的融合表达载体pGEX-rhGLP-1类似物，转化大肠杆菌BL21（DE3）获得重组菌株。IPTG诱导表达的菌体经高压均质机破碎后，离心收集包涵体，经尿素变性、Glutathione-Sepharose 4B亲和层析、肠激酶酶切、SP-Sepharose FF层析和反相层析RP-C18脱盐后冻干，得到纯度大于96%的rhGLP-1类似物，经质谱测定，分子量与理论值一致。生物学活性分析表明，rhGLP-1类似物具有促进表达有GLP-1受体的HEK293细胞cAMP增加的活性。
关键词： 重组人胰高血糖素样肽-1;  生物学活性;  纯化;  融合蛋白    
Abstract:

The synthetic recombinant human glucagon-like peptide-1(7~37) (rhGLP-1) analogue gene was cloned into prokaryotic expressing vector pGEX-4T-3 fused with glutathione-S-transferases (GST) to construct a recombinant plasmid pGEX-rhGLP-1 analogue, then transformed into E. coli BL21(DE3). The constructed E. coli BL21(DE3)/pGEX-rhGLP-1 analogue was induced by IPTG and proteins were expressed, and then broken to extract inclusion bodies by high pressure homogenizer. The inclusion bodies were collected through centrifugation. The rhGLP-1 analogue was purified by urea denaturation, Glutathione-Sepharose 4B affinity chromatography and digested by enterokinase. By further purification with SP-Sepharose FF chromatography and RP-C18 reverse phase chromatography, the target rhGLP-1 analogue was gained with purity more than 96% by HPLC analysis and molecular weight was consistent with theoretical value by MS analysis. The biological activity analysis showed that rhGLP-1 analogue stimulated cAMP production from HEK293 cells expressing GLP-1 receptors.
Key words: Recombinant human glucagon like peptide -1    Fusion protein    Purification    Biological activity
收稿日期: 2016-06-03 出版日期: 2016-12-25
ZTFLH:  Q819  
基金资助:

广东省引进创新科研团队计划资助项目（201101Y0104990178）
通讯作者: 陈英     E-mail: chenying@hec.cn
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引用本文:

高相雷, 林树珊, 龚庆伟, 潘兰, 马利, 冯艳, 林小鹊, 曾剑, 李文佳, 陈小锋, 陈英. 重组人胰高血糖素样肽-1类似物的分离纯化和鉴定[J]. 中国生物工程杂志, 2016, 36(12): 15-20.

GAO Xiang-lei, LIN Shu-shan, GONG Qing-wei, PAN Lan, MA Li, FENG Yan, LIN Xiao-que, ZENG Jian, LI Wen-jia, CHEN Xiao-feng, CHEN Ying. Purification and Characterization of Recombinant Human Glucagon-like Peptide-1 Analogue. China Biotechnology, 2016, 36(12): 15-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/DOI:10.13523/j.cb.20161203    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I12/15

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