The gene coding for the STb enterotoxin will be inserted into a plasmid vector and cloned into a K12 strain of E coli. The clones will be screened by DNA hybridization, immunoassay and bioassay to select strains which hyperproduce the STb toxin. Purification of the toxin will be done on culture supernatants using standard chromatography, an FPLC unit and antigen-antibody affinity columns. The purified toxin will then be biochemically analyzed. Antibodies to the STb toxin will be made by coupling purified or synthetic toxin to a large molecule and injecting the coupled protein into rabbits. The antibodies will be used in an immunoassay, and antigen-antibody affinity column and neutralization an receptor site studies. The gene for CJT toxin production will be localized on plasmid or chromosomal DNA. A genomic library will be created in pUC or lambda phage and screened for toxin production byh bioassay, Y1 cell assay and immunoassay. Once the gene for CJT production is identified it will be sequenced and then cloned into an expression vector. The cjt pUC clones will be transferred into K12 E. coli and selected for hyperproduction of C. jejuni enterotoxin. The toxin will be purified by standard chromatography methods for use in future studies.
