Coloma M J, Hastings A, Wims L A, Morrison S L
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
J Immunol Methods. 1992 Jul 31;152(1):89-104. doi: 10.1016/0022-1759(92)90092-8.
A new family of vectors has been produced which facilitates the cloning and expression of immunoglobulin variable regions cloned by polymerase chain reaction (PCR). The vectors are designed to express the cloned variable regions joined to human constant regions and take advantage of priming in the leader sequence so that no amino acid changes will be introduced into the mature antibody molecule. Both the heavy chain and light chain vectors utilize a murine VH promoter provided with an EcoRV restriction site so that the amplified variable regions can be directly cloned into a functional promoter. For the heavy chain an NheI restriction site has been generated at the first two amino acids of CH1 and the cloned leader and variable region are fused directly to the CH1 domain of the constant region. When the leader and variable regions of the light chain were fused directly to C kappa, no expression was observed. Therefore the light chain expression vector was designed with a SalI restriction site for cloning into a splice junction 3' of the variable region; VL then is joined to C kappa by splicing. Both vectors direct the expression of functional, fully assembled immunoglobulin molecules with the expected molecular weight. Use of redundant oligomers to prime the PCR permits the cloning and expression of recombinant antibodies without any prior information as to their sequence and makes it possible to rapidly generate recombinant antibodies from any monoclonal antibody producing cell line.
