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    程露, 易小萍, 孙祥明, 张元兴. 鼠源抗过敏融合蛋白的瞬时表达及纯化[J]. 华东理工大学学报(自然科学版), 2011, (5): 532-537.
    引用本文: 程露, 易小萍, 孙祥明, 张元兴. 鼠源抗过敏融合蛋白的瞬时表达及纯化[J]. 华东理工大学学报(自然科学版), 2011, (5): 532-537.
    CHENG Lu, YI Xiao-ping, SUN Xiang-ming, ZHANG Yuan-xing. Transient Gene Expression and Purification of a MouseDerived AntiAllergy Fusion Protein[J]. Journal of East China University of Science and Technology, 2011, (5): 532-537.
    Citation: CHENG Lu, YI Xiao-ping, SUN Xiang-ming, ZHANG Yuan-xing. Transient Gene Expression and Purification of a MouseDerived AntiAllergy Fusion Protein[J]. Journal of East China University of Science and Technology, 2011, (5): 532-537.

    鼠源抗过敏融合蛋白的瞬时表达及纯化

    Transient Gene Expression and Purification of a MouseDerived AntiAllergy Fusion Protein

    • 摘要: 采用流加和降温培养工艺,实现在5 L搅拌式生物反应器规模的瞬时基因表达。首先比较了两种表达载体在CHOS细胞中的瞬时基因表达,发现pIDmAAFP载体介导的瞬时转染表达可以获得mAAFP的高水平表达。采用pIDmAAFP载体在1.3 L搅拌式生物反应器中进行瞬时基因表达,与分批培养相比,通过流加和降温培养工艺对转染过程进行控制,培养时间延长1倍,蛋白表达浓度提高8倍。在5 L搅拌式生物反应器中对上述工艺进行成功放大,mAAFP质量浓度达到25 mg/L。之后,利用rProtein A Sepharose Fast Flow介质纯化mAAFP,并通过SDSPAGE、Western blot验证其纯度,为下一步利用小鼠模型研究蛋白功效和作用机理做好准备。

       

      Abstract: To improve the transient gene expression of a mousederived antiallergy fusion protein (mAAFP) in 5 L bioreactor, feeding and mild hypothermia were adopted together to provide sufficient nutrition for cell growth and extend the production duration. Firstly, two different expression vectors of pCImAAFP and pIDmAAFP were transiently transfected in CHOS cells with an optimal transient transfection protocol and a better level of mAAFP expression was reached when using pIDmAAFP as an expression vector. Transient gene expression (TGE) was conducted in 1.3 L bioreactor, with combined use of fedbatch and mild hypothermic culture, so the expression level of mAAFP was increased 8fold compared with batch culture. Finally, the TGE process was carried out in a 5 L bioreactor and a high expression level of 25 mg/L was achieved. mAAFP was subsequently purified by rProteinA Sepharose Fast Flow. The obtained mAAFP was identified by SDSPAGE and Western blot. This work provided the necessary basis to assay mAAFP efficacy in mouse later on.

       

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