利用抗凋亡CHO细胞株BAK-/BAX-制备重组胰高血糖素样肽-1与人血清白蛋白融合蛋白GGH
首发时间:2020-10-10
摘要:目的 以 BAK-/BAX- 双敲除的抗凋亡 CHO 细胞株为宿主,制备N端整齐、安全性高的重组胰高血糖素样肽-1 (GLP-1) 与人血清白蛋白 (HAS) 融合蛋白(GGH)。方法 构建重组质粒pcDNA3.1/GGH,采用脂质体转染法将重组质粒转染抗凋亡的 CHO 细胞株,通过 G418 压筛,有限稀释法挑选单克隆,Dot-Blot 和 Western-Blot 进行验证,筛选出稳定表达 GGH 的细胞株;利用该细胞株进行 3L 摇瓶流加发酵培养,用 Blue Sepharose 6 Fast Flow 和 Phenyl Sepharose High Performance 进行纯化,并将纯化产物在 CHO-S-GLP-1R-EGFP 细胞上通过检测 cAMP 的生成进行初步的活性分析。结果 构建及筛选出能稳定表达 GGH 的抗凋亡 CHO 细胞株;将该细胞株进行 3L 摇瓶流加培养,产量达到 163mg/L;纯化后产物有良好的生物学活性。结论 成功构建及筛选了表达 GGH 的抗凋亡 CHO 细胞株,该细胞株在进行 3L 摇瓶流加培养后产量达到 163mg/L,纯化产物展现出良好的生物学活性,为今后的研究奠定了基础。
关键词: GGH CHO细胞 转染 流加培养 分离纯化 活性
For information in English, please click here
Preparation of fusion protein GGH of glucagon-like peptid-1 and human serum albumin with anti-apoptotic BAK-/BAX- CHO cell
Abstract:Objective: Prepare the N-terminal consistent and high-safe recombinant glucagon-like peptide-1 (GLP-1) and human serum albumin (HSA) fusion protein (GGH) with the BAK/BAXdouble knockout anti-apoptotic CHO cells. Methods: Construct the recombinant plasmid pcDNA3.1/GGH, then transfect it into the anti-apoptotic CHO cells with liposome transfection method. Select single clones through G418 pressure selectionand limited dilution method. Cell lines stably expressing GGH were selected through Dot-Blot and Western-Blot. The highest expression cell clone was cultured in fed-batch in 3L shake flask. The fermentation broth is purified with Blue Sepharose 6 Fast Flow and Phenyl Sepharose High Performance. The activity of fusion protein GGH was analyzed on CHO-S-GLP-1R-EGFP cells to detect cAMP production. Results: The anti-apoptotic CHO cell line that can stably express GGH was constructed and screened. The cell line was cultured in a 3L shake flask and the yield reached 163mg/L and the purified product had good biological activity. Conclusion: Successfully constructed and screened an anti-apoptotic CHO cell line expressing GGH. The yield of the cell line reached 163 mg/L after 3L shake flask cultured and the purified product showed good biological activity, which laid the foundation for future research.
Keywords: GGH;CHO cells;Transfection; Fed-batch culture; Separation and purification;Activity
基金:
引用
No.****
动态公开评议
共计0人参与
勘误表
利用抗凋亡CHO细胞株BAK-/BAX-制备重组胰高血糖素样肽-1与人血清白蛋白融合蛋白GGH
评论
全部评论0/1000