Convenient plasmid vectors for construction of chimeric mouse/human antibodies.
Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and V kappa genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with JH and J kappa probes, respectively, and such fragments can be isolated in lambda-EcoRI and lambda-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG 1 gpt contains human C gamma 1 and Ecogpt genes, and only one EcoRI site upstream of the C gamma 1 gene; pSV2-HC kappa neo contains human C kappa and neo genes, and only one HindIII site upstream of the C kappa gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a V kappa J kappa gene are inserted into pSV2-HC kappa neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.
Kameyama
K
K
Institute for Comprehensive Medical Science, Fujita-Gakuen Health University, Aichi, Japan.
Imai
K
K
Itoh
T
T
Taniguchi
M
M
Miura
K
K
Kurosawa
Y
Y
D000818:Animals; D002678:Chimera; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D015183:Restriction Mapping
D007127:Immunoglobulin Constant Regions; D007135:Immunoglobulin Variable Region