GLP-1-Fc融合蛋白在DG44细胞中的表达及其质量分析

发布时间：2019-04-04 16:27

【摘要】：构建GLP-1-Fc蛋白真核表达载体,并在DG44细胞株中实现表达。利用基因工程等方法构建表达GLP-1-Fc融合蛋白的重组DG44细胞株,通过甲氨喋呤的加压筛选及有限稀释法挑选出表达量较高的单克隆细胞株,经无血清驯化培养获得悬浮生长的细胞株。收集发酵培养上清,通过离心、过滤及亲和层析等方法对目的蛋白GLP-1-Fc进行分离纯化,并利用Dot blot、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效液相色谱法、Edman降解等方法进行目的蛋白的质量分析,以建立GLP-1-Fc融合蛋白发酵、纯化工艺,探索该融合蛋白的质量检测方法。本实验成功构建了表达GLP-1-Fc融合蛋白的重组DG44悬浮型细胞株,通过该工艺流程,GLP-1-Fc融合蛋白表达量达到400 mg/L,纯度达95%,分子量约为3.0×10~4。该生产工艺能够获得与理论目标蛋白相一致的、且结构稳定GLP-1-Fc融合蛋白,其质量检测方法稳定可靠。
[Abstract]:The eukaryotic expression vector of GLP-1-Fc protein was constructed and expressed in DG44 cell line. The recombinant DG44 cell line expressing GLP-1-Fc fusion protein was constructed by genetic engineering, and the monoclonal cell lines with high expression were selected by pressure screening of methotrexate and limited dilution method. The suspension growth cell lines were obtained by serum-free acclimation culture. The supernatant of fermentation culture was collected and purified by centrifugation, filtration and affinity chromatography. The target protein GLP-1-Fc was separated and purified by Dot blot, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography (HPLC). Edman degradation and other methods were used to analyze the quality of the target protein in order to establish the fermentation and purification process of the GLP-1-Fc fusion protein and to explore the quality detection method of the fusion protein. The recombinant DG44 suspension cell line expressing GLP-1-Fc fusion protein was successfully constructed in this experiment. Through this process, the purity of GLP-1-Fc fusion protein was up to 400 mg/L, and the molecular weight was about 3.0 脳 10 ~ 4. The purity of GLP-1-Fc fusion protein reached 95% and the molecular weight was about 3.0 脳 10 ~ 4. The GLP-1-Fc fusion protein with stable structure and consistent with the theoretical target protein can be obtained by this process, and its quality detection method is stable and reliable.
【作者单位】：南京工业大学生物与制药工程学院;上海凯茂生物医药有限公司;南京汇科生物工程设备有限公司;
【基金】：国家高技术研究发展计划(863计划)(2012AA021201)
【分类号】：R915

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