GLP-1-IgG4-Fc融合蛋白的表达与鉴定 <sup>*</sup>

doi:10.13523/j.cb.20180708

中国生物工程杂志

2018, Vol. 38

Issue (7): 58-66 DOI: 10.13523/j.cb.20180708

技术与方法

GLP-1-IgG4-Fc融合蛋白的表达与鉴定 ^*

陈英(

),肖海鹏,张晓焰,龚庆伟,马利,李文佳,陈小锋

广东东阳光药业有限公司东莞 523867

Expression and Characterization of Recombinant GLP-1-IgG4-Fc Fusion Protein

Ying CHEN(

),Hai-peng XIAO,Xiao-yan ZHANG,Qing-wei GONG,Li MA,Wen-jia LI,Xiao-feng CHEN

HEC Pharma Co. Ltd, Guangdong, Dongguan 523867, China

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摘要：

将合成的人胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)突变体基因与IgG4抗体的Fc部分进行融合获得GLP-1-IgG4-Fc片段,获得的基因片段与pXC17.4载体进行连接,用电转化方法将线性化质粒稳定转染CHO-K1细胞,通过ClonePix 2筛选出高表达细胞株,产量达1.5g/L。收集培养上清并经Protein A和Source 30Q纯化,得到的GLP-1-IgG4-Fc融合蛋白,SDS-PAGE纯度高于95%,高效液相色谱(high performance liquid chromatography,HPLC)纯度和毛细管区域凝胶电泳(capillary zone electrophoresis,CZE)纯度均不低于80%,尺寸排阻层析(size-exclusion chromatography,SEC)纯度高于99%。经质谱和肽图谱测定,分子量与理论值一致,肽图谱序列与对照品高度一致。生物学活性分析表明,GLP-1-IgG4-Fc融合蛋白具有促进表达有GLP-1受体的HEK293细胞分泌环磷酸腺苷(cyclic adenosine monophosphate,cAMP)的活性,并且该活性与对照品高度相似。

关键词： 重组人胰高血糖素样肽-1; GLP-1-IgG4-Fc融合蛋白; CHO细胞; 生物学活性

Abstract:

The GLP-1 mutant gene and Fc of IgG4 were fused into GLP-1-IgG4-Fc, and were inserted into a mammalian expression vector pXC17.4. CHO-K1 cell was stable transfected with linearlized plasmid.High expressing clone with yield of 1.5g/L was got through ClonePix 2. The target GLP-1-IgG4-Fc was gained with SDS-PAGE purity more than 95% after purification by Protein A and Source 30Q. The HPLC and CZE purity of the target protein were more than 80%, and the purity was more than 99% by SEC analysis. The molecular weight was consistent with the theoretical value by MS and the peptide map was the same as that of the control by peptide mapping analysis. The biological activity analysis showed that GLP-1-IgG4-Fc stimulated cAMP production in HEK293 cell expressing GLP-1 receptors, and the biological activity was highly similar to the control.

Key words: Recombinant human glucagon like peptide-1 GLP-1-IgG4-Fc fusion protein CHO cell Biological activity

收稿日期: 2017-12-19 出版日期: 2018-08-13

ZTFLH:

Q78

基金资助: 广东省引进创新科研团队计划(201101Y0104990178)

通讯作者: 陈英 E-mail: chenying@hec.cn

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引用本文:

陈英,肖海鹏,张晓焰,龚庆伟,马利,李文佳,陈小锋. GLP-1-IgG4-Fc融合蛋白的表达与鉴定 ^*[J]. 中国生物工程杂志, 2018, 38(7): 58-66.

Ying CHEN,Hai-peng XIAO,Xiao-yan ZHANG,Qing-wei GONG,Li MA,Wen-jia LI,Xiao-feng CHEN. Expression and Characterization of Recombinant GLP-1-IgG4-Fc Fusion Protein. China Biotechnology, 2018, 38(7): 58-66.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180708 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/58

图1 质粒pXC17.4和pUC-57-GLP-1-IgG4-Fc酶切示意图

图2 pXC17.4-GLP-1-IgG4-Fc酶切示意图

图3 pXC17.4-GLP-1-IgG4质粒图谱

图4 细胞Pools的Clone Select Image成像示意图

图5 克隆半固体培养后的ClonePix 2成像示意图

图6 Source 30Q层析纯化GLP-1-IgG4-Fc样品RP-HPLC分析图

图7 Source 30Q层析纯化GLP-1-IgG4-Fc样品CZE分析图

图8 Source 30Q层析纯化GLP-1-IgG4-Fc样品SEC分析图

图9 GLP-1-IgG4-Fc融合蛋白纯化后的SDS-PAGE分析图谱

图10 质谱测定GLP-1-IgG4-Fc的分子量

图11 GLP-1-IgG4-Fc的肽图谱分析

图12 GLP-1-IgG4-Fc的体外生物活性测定

表1 对照品与rGLP-1半数效应对比

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