The expression and characterization of a series of rate IgE produced from in vitro reconstructed epsilon heavy chain genes
McMillan, Daniel Randy
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The interaction between allergen-specific IgE and the high affinity Fc receptor on the surface of mast cells or tissue basophils forms the basis for the Type I Immediate Hypersensitivity reaction, or more commonly, the allergic response. In order to rigorously analyze this interaction, a series of exon modules have been produced to facilitate the production of mutant IgE. The functionally rearranged e-heavy chain gene isolated from the rat IgE secreting immunocytoma, IR162, was utilized for the creation of this system of exon modules. The five individual exons, encoding the variable and constant region domains, were isolated and subcloned into the multiple cloning site of a pair of plasmid vectors in such a way as to preserve or reconstruct all the required mRNA splicing, cleavage and polyadenylation signals. The vectors utilized here permit the use of flanking restriction enzyme sites for the modular manipulation of the e-heavy chain gene. These exon modules were initially used to reconstruct the e-heavy chain gene into the native configuration to demonstrate the efficacy of the modular system for synthesis of IgE. Upon transfection into the rat myeloma cell line, Y3, the reconstructed gene produced a polypeptide that associated with the endogenous light chain polypeptide and was secreted from the cell. The immunoglobulin was indistinguishable fix)m authentic IgE, based on heavy chain specific mRNA initiation, overall e-heavy chain specific mRNA length, size of polypeptides recognized by anti-IgE antibodies and high affinity Fc epsilon receptor binding activity. The modular system of exons was then used to produce mutant IgE that were deficient in one or more whole domain(s) through the systematic assembly of different mutant e-heavy chain gene constructions. The data obtained with the full-length eheavy chain gene validates the efficacy of the modular approach and suggests that the use of mutant IgE, produced in such a manner, in receptor binding studies will provide information on the specific domain(s) involved in interaction with the receptor. In addition, a chimeric x-light chain gene coding for a variable region that contributes to specificity for the hapten, trinitrophenol, was produced and used to develop a second host cell line. Utilization of this cell line will permit the production of recombinant IgE with known antigen specificity for further purification and functional studies. Construction of the exon modules, full-length and mutant e-heavy chain genes and the physical and functional characterizations of the resulting RNA and polypeptide products of the full-length and mutant e-heavy chain genes are described.