Expression of rabbit IgA heavy chain genes in E. coli and in murine myeloma cells.
Rabbit IgA-heavy chain cDNA and germline genes were cloned into prokaryotic and eukaryotic expression vectors, respectively. The Fc alpha encoding portion of six C alpha cDNA clones were cloned into pUC8 and E. coli were transformed. Radioimmunoassay of the molecules synthesized by these clones showed that molecules with Fc alpha antigenic determinants were produced at the level of approximately 0.1 to 1.0 microgram per ml culture. Radiobinding analysis showed that each of the clones encoded heavy chains of the IgA-g subclass. Southern blot analysis of rabbit germline DNA revealed 10 germline C alpha genes. Five of these, isolated from recombinant cosmid libraries, were cloned into a eukaryotic expression vector containing a rearranged murine VDJ gene, the CH enhancer region and the Eco-gpt gene. Murine myeloma cells, J558L, were transfected with each of the heavy chain constructs and stable transfectants was selected with mycophenolic acid. The immunoglobulins produced by each transfectant were analyzed by radiobinding and by SDS-PAGE. Each transfectant were shown to synthesize IgA molecules and thus all five C alpha genes are expressible. The heavy chains from the transfectants ranged in size from 55,000 to 60,000 daltons. Radiobinding analyses indicated that four of the five genes encode molecules of the IgA-f subclass; the serological identity of the fifth gene is not yet established.
D000818:Animals; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007070:Immunoglobulin A / Q000235:genetics*; D007126:Immunoglobulin Allotypes / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D009101:Multiple Myeloma / Q000473:pathology; D011817:Rabbits / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D007070:Immunoglobulin A; D007126:Immunoglobulin Allotypes; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA