Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG1, IgG2, and IgG3 molecules.
A bovine recombinant phage library was constructed and screened with rabbit S mu and human C gamma probes. IgM, IgA, and IgE H chain constant region (CH) genes were isolated with the rabbit S mu probe and three IgG CH genes were isolated with the C gamma probe. The CH genes were individually cloned into an expression vector which contained a murine VDJ gene cloned from a hybridoma producing anti-dansyl hapten antibody. The resulting constructs were transfected into murine hybridoma cells producing L chain of the anti-dansyl antibody and stable transfectomas secreting chimeric bovine-murine IgA, IgE, or IgG subclass anti-dansyl antibodies were obtained. The chimeric antibodies, immunoprecipitated with Ag or with anti-bovine H chain antibodies, were analyzed by SDS-PAGE and were shown to contain H and L chains of expected size. Of the three chimeric antibodies derived from the C gamma genes, one reacted with anti-IgG1 antibody, another reacted with anti-IgG2 antibody and the third did not react with either anti-IgG1 or anti-IgG2. This third IgG appears to represent a "new" subclass of bovine IgG, IgG3. Southern blot analysis indicated that the bovine genome contains a fourth C gamma gene. These experiments demonstrate the usefulness of molecular genetic techniques for the isolation and characterization of Ig which are not readily purified from biologic fluids. These techniques will be useful for isolation and characterization of Ig genes from other outbred mammals.
Knight
K L
KL
Department of Microbiology and Immunology, University of Illinois, Chicago 60612.
Suter
M
M
Becker
R S
RS
D000818:Animals; D002417:Cattle / Q000235:genetics* / Q000276:immunology; D002678:Chimera; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D005803:Genes, Immunoglobulin; D005854:Germ Cells / Q000032:analysis; D006241:Haptens / Q000235:genetics; D007070:Immunoglobulin A / Q000235:genetics; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007073:Immunoglobulin E / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D007136:Immunoglobulins / Q000235:genetics*; D008297:Male; D051379:Mice; D011485:Protein Binding
D006241:Haptens; D007070:Immunoglobulin A; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007136:Immunoglobulins; D007073:Immunoglobulin E; D004247:DNA