Abstract
We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.
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Acknowledgments
We thank Prof. Guo Yajun, International Joint Cancer Institute, The Second Military Medical University, for supplying vector pcDNA3-CH and pcDNA3-CL.
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This study was supported by the National Key Basic Research Program (973 Program) funded project (2010CB833605), People’s Republic of China.
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Li, F., Liu, YH., Li, YW. et al. Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Appl Microbiol Biotechnol 96, 1233–1241 (2012). https://doi.org/10.1007/s00253-012-4243-5
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DOI: https://doi.org/10.1007/s00253-012-4243-5