文献类型：期刊文献

中文题名：鼠源抗过敏融合蛋白的瞬时表达及纯化

英文题名：Transient Gene Expression and Purification of a Mouse-Derived Anti-Allergy Fusion Protein

作者：程露[1];易小萍[1];孙祥明[2];张元兴[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]上海富莼科芯生物技术有限公司,上海201203

年份：2011

卷号：37

期号：5

起止页码：532

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

基金：国家反应器重点实验室开放课题基金(2009ZX09503-013-2);国家科技重大专项(2009ZX09102-249)

语种：中文

中文关键词：融合蛋白;瞬时基因表达;中国仓鼠卵巢细胞;过敏

外文关键词：fusion protein; transient gene expression; chinese hamster ovary（CHO） cells; allergy

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。
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.