Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells

J Immunol Methods. 1997 May 12;204(1):77-87. doi: 10.1016/s0022-1759(97)00034-3.

Abstract

We have developed new cassette expression vectors for the cloning of any intact V-region gene followed by any C-region gene. Both the heavy-and light chain vectors harbor a strong hCMV promoter, restriction site cassettes for cloning of both V- and C-region genes, transcription termination signals, fl-ori for single stranded DNA (ssDNA) synthesis, selection marker for Neomycin and SV40 ori for transient expression. The vectors accept VH and VL chain genes obtained by RT-PCR. Reamplification of the V genes is then performed with a new set of primers which are designed specifically for each individual V gene. Cloning into the vectors is aided by restriction sites located just outside the V-gene coding region, thus keeping the V-genes intact. The vectors also contain cloning sites for the exchange of genomic C-genes so that the resulting Ig genes may code for complete antibodies, antibody fragments or fusion proteins. A simple subcloning step permits the expression of both heavy and light chain genes from one single vector, thus avoiding co-transfection of the two vectors. The usefulness of the vectors was confirmed by construction of mouse-human chimeric antibodies. The V-genes were derived from a hybridoma cell line, TP-3, and was combined with human C kappa, C gamma 3 and C gamma 1 genes as well as with CH1 gamma 3. High yields of recombinant antibody products in NSO cells were obtained. Transient expression was also demonstrated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • COS Cells
  • Cloning, Molecular
  • Gene Expression
  • Genetic Vectors*
  • Humans
  • Hybridomas
  • Immunoglobulin Fab Fragments / immunology
  • Immunoglobulin Heavy Chains / genetics*
  • Immunoglobulin Variable Region / genetics*
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Fab Fragments
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region
  • Recombinant Fusion Proteins