Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma

Appl Microbiol Biotechnol. 2012 Dec;96(5):1233-41. doi: 10.1007/s00253-012-4243-5. Epub 2012 Jul 7.

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.

MeSH terms

  • Antibodies, Neoplasm / genetics
  • Antibodies, Neoplasm / immunology*
  • Antibodies, Neoplasm / isolation & purification
  • Carcinoma, Hepatocellular / immunology*
  • Carcinoma, Hepatocellular / pathology*
  • Cell Line
  • Cell Surface Display Techniques / methods
  • Gene Expression
  • Genetic Vectors
  • Humans
  • Immunoglobulin G / genetics
  • Immunoglobulin G / immunology
  • Immunoglobulin G / isolation & purification
  • Peptide Library*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification

Substances

  • Antibodies, Neoplasm
  • Immunoglobulin G
  • Peptide Library
  • Recombinant Proteins