A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies
A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies
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DOI：
10.1016/j.jim.2006.10.010
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发表时间：
2007-01-10
影响因子：
2.2
通讯作者：
Jostock, Thomas
中科院分区：
文献类型：
--
作者：
Li, Jiandong;Menzel, Christian;Jostock, Thomas
单克隆抗体（Mab）是发展中增长最快的生物制药组。为了在哺乳动物细胞中产生，免疫球蛋白双异源四聚体的四条多肽链必须在离开内质网之前组装。利用在一个或两个质粒上编码的单顺反子和双顺反子表达盒，已经开发了各种重组Mab表达载体。然而，只有少数研究提供了关于稳定或瞬时生产重组IgG的最佳载体设计类型的信息。因此，在本研究中，我们构建了一系列哺乳动物表达载体，用于生产具有不同表达盒设计的重组人或嵌合IgG抗体。产生了具有不同启动子和顺反子排列的单顺反子和双顺反子表达的版本。在瞬时转染的293 T和CHO-K1细胞中评价抗体产生水平。此外，生成稳定的CHO细胞系并分析抗体生产水平和稳定性。我们的结果表明，与单顺反子表达相比，EMCV IRES介导的双顺反子表达构建体产生相似的抗体表达水平，并在CHO细胞系中显示出长期稳定性。添加编码YFP的第三顺反子显示出有利于使用FACS分选仪筛选和分离克隆。(c)2006 Elsevier B.V保留所有权利。
Monoclonal antibodies (Mab) are the fastest growing group of biopharmaccuticals in development. For production in mammalian cells, the four polypeptide chains of the immunoglobulin diheterotetramer must be assembled prior to exit from the endoplasmic reticulum. Various recombinant Mab expression vectors have been developed utilizing mono-and bicistronic expression cassettes encoded on one or two plasmids. However, there are only few studies providing information on the type of vector design optimal for stable or transient production of recombinant IgG. Consequently, in this study, we have constructed a series of mammalian expression vectors for the production of recombinant human or chimeric IgG antibodies with different expression cassette designs. Versions for monocistronic and bicistronic expression with different promoters and cistron arrangements were generated. Antibody production levels were evaluated in transiently transfected 293T and CHO-K1 cells. Furthermore, stable CHO cell lines were generated and analyzed for antibody production levels and stability. Our results indicate that compared to monocistronic expression, EMCV IRES-mediated bicistronic expression constructs yield similar antibody expression levels and show long-term stability in CHO cell lines. Addition of a third cistron encoding YFP was shown to facilitate screening and isolation of clones using a FACS sorter. (c) 2006 Elsevier B.V All rights reserved.