Immunopotentiation of Mucosal Secretory IgA Responses by Use of a Ganglioside Binding Probe

Abstract

Abstract : In order to facilitate the production of the B subunit of the heat- labile enterotoxin of E. coli (LT-B) for use as an immunologic carrier, we utilized recombinant DNA technology to construct a plasmid containing the genes for production of the B subunit only from a human isolate of enterotoxigenic E. coli. the 0.8kb gene fragment encoding synthesis of LT-B was cloned into plasmid pBR322 following sequential digestion of the enterotoxin plasmid with restriction endonucleases PstI and HindIII. Cloned B subunit was isolated from cell lysates in its oligomeric form, was structurally identical to native B subunit when examined by SDS-PAGE, immunologically identical in ELISA, and contained no demonstrable A subunit in either assay. Depsite deletion of the A subunit, cloned LT-B did induce morphologic alterations in cultured mouse Y1 adrenal cells at high concentrations, an indication of residual toxicity. The 0. 8kb gene fragment was recloned into the plasmid vector pUC8 and this resulted in a construct which coded for synthesis of LT-B free of biologic activity but still capable of binding to the epithelial cell surface. Peptide fragmentation resulted in loss of binding activity, suggesting a conformational binding determinant.