TREATMENT FOR VEROTOXIN-PRODUCING ESCHERICHIA COLI

Abstract: The present invention includes methods for generating neutralizing antitoxin directed against verotoxins. In particular, the antitoxin directed against these toxins is produced in avian species using soluble recombinant verotoxin proteins. This avian antitoxin is designed so as to be administrable in therapeutic amounts and may be in any form (i.e., as a solid or in aqueous solution). These antitoxins are useful in the treatment of humans and other animals intoxicated with at least one bacterial toxin, as well as for diagnostic assays to detect the presence of toxin in a sample.

Description: TREATMENT FOR VEROTOXIN-PRODUCING ESCHERICHIA COLI FIELD OF THE INVENTION The present invention relates to antitoxin therapy for humans and other animals, and diagnostic assays to detect toxins. Antitoxins which neutralize the pathologic effects of Escherichia coli toxins, such as verotoxin are provided. BACKGROUND OF THE INVENTION A. Escherichia coli as a Pathogenic Organism Escherichia cυli is the organism most commonly isolated in clinical microbiology laboratories, as it is usually present as normal flora in the intestines of humans and other animals. However, it is an important cause of intestinal, as well as extraintestinal infections. For example, in a 1984 survey of nosocomial infections in the United States. E. coli was associated with 30.7% of the urinary tract infections. 1 1.5% of the surgical wound infections. 6.4% of the lower respiratory tract infections. 10.5% of the primary bacteremia cases. 7.0% of the cutaneous infections, and 7.4% of the other infections (J.J. Farmer and M.T. Kelly. "Enterobacteriaceae." in Manual of Clinical Microbiology. Balows et _./.(eds). American Society for Microbiology, [1991], p. 365). Surveillance reports from England. Wales and Ireland for 1986 indicate that E. coli was responsible for 5.473 cases of bacteremia (including blood, bone marrow, spleen and heart specimens): of these. 568 were fatal. For spinal fluid specimens, there were 58 cases, with 10 fatalities (J.J. Farmer and M.T. . "Enterobacteriaceae." in Manual of Clinical Microbiology. Balows ei _//.(eds). American Society for Microbiology, [ 1991 ], p. 366 ). There are no similar data for United States, as these are not reportable diseases in this country. Studies in various countries have identified certain serotypes (based on both the O and H antigens) that are associated with the four major groups of E. coli recognized as enteric pathogens. Table 1 lists common serotypes included within these groups. The first group includes the classical enteropathogenic serotypes ("EPEC"): the next group includes those that produce heat-labile or heat-stable enterotoxins ("ETEC"): the third group includes the enteroinvasive strains ("EIEC") that mimic Shigella strains in their ability to invade and multiply within intestinal epithelial cells: and the fourth group includes strains and serotypes that cause hemorrhagic colitis or produce Shiga-like toxins (or verotoxins) ("VTEC" or "EHEC" [enterohemmorrhagic E. coli]). Table 1. Pathogenic E. coli Serotypes Group Associated Serotypes Enterotoxigenic O6:H16: O8:NM; O8:H9; Oll:H27: O15:Hll: O20.NM: O25:NM; (ETEC) O25:H42: O27:H7: O27:H20: O63:H12; O78:Hll: O78:H12; O85:H7; O114:H21: O115:H21; O126:H9: O128ac:H7: O128ac:H12: O128ac:H21; O148:H28: 0149.H4: O159.H4: O159:H20: O166:H27; and O167:H5 Enteropathogenic O26:NM: O26:Hll; O55:NM: O55:H6: O86:NM: O86:H2: (EPEC) O86:H34: Olllab:NM; Olllab:H2: Olllab:H12: Olllab:H21: O114:H2: O119:H6: O125ac:H21: O127.NM: O127:H6: O127:H9: O127:H21: O128ab:H2: O142:H6: and O158:H23 Enteroinvasive O28ac:NM: O29:NM; O112ac:NM: O115:NM: 0124:NM: (EIEC) O124:H7: O124.Η30: O135:NM: O136:NM: 0143:NM: 0144:NM: O152:NM: O164:NM: and O167:NM Verotoxin-Producing Ol:NM: O2:H5: O2:H7: O4:NM: O4:H10: O5:NM: O5:H16: (VTEC)) O6:Hl: O18:NM; O18:H7; O25:NM: 026:NM: 026:H11: O26:H32: O38:H21: O39:H4: O45:H2: O50:H7: 055.H7: O55:H10: O82:H8; O84:H2: O91:NM: O91:H21: O103:H2: Olll.NM; Olll:H8: O111:H30; Olll:H34; O113:H7: OI13:H21: O114:H48: O115.H10; O117:H4; O118:H12: O118:H30: 0121:NM: O121:H19: O125:NM: O125:H8; O126:NM; O126:H8: O128:NM: O128:H2: O128:H8: O128:H12: O128:H25: O145:NM: O125:H25: O146:H21: O153:H25: O157:NM; O157:H7: O163:H19: O165:NM: 0165:19: and O165:H25 B. Verotoxin Producing Strains of E. coli Although all of these disease-associated serotypes cause potentialh life-threatening disease. E. coli O157:H7 and other verotox in-producing strains have recently gained widespread public attention in the United States due to their recently recognized association with two serious extraintestinal diseases, hemolvtic uremic syndrome ("HUS") and thrombotic thrombocytopenic purpura ("TTP"). Worldwide. E. coli O157:H7 and other verotoxin- producing E. coli (VTEC) are an increasingly important human health problem. First identified as a cause of human illness in early 1982 following two outbreaks of food-related hemorrhagic colitis in Oregon and Michigan (M.A. Karmali. "Infection by Verocytotoxin- Producing Escherichia coli." Clin. Microbiol. Rev..2:15-38 [1989]: and L. W. Riley. ci al. "Hemorrhaeic colitis associated with a rare Escherichia coli scrotype." New Eng. J. Med.. i . 308: 681-685 [1983]). the reported incidence of VTEC-associated disease has risen steadily, with outbreaks occurring in the U.S.. Canada, and Europe. With increased surveillance. £ coli O157.Η7 has been recognized in other areas of the world including Mexico. China. Argentina. Belgium, and Thailand (N. V. Padhye and M. P. Doyle. "Escherichia coli O157:H7: Epidemiology, pathogenesis and methods for detection in food." J. Food. Prot.. 55: 555-565 [1992]: and P. M. Griffin and R. V. Tauxe. "The epidemiology of infections caused by Escherichia coli O157:H7. other enterohemorrhagic £. coli. and the associated hemolvtic uremic syndrome." Epidemiol. Rev.. 13: 60 [ 1991 ]). The disease attracted national attention in the U.S. after a major outbreak in the Pacific Northwest that was associated with consumption of undercooked E. coli O157.Η7- contaminated hamburgers. Over 700 hundred people fell ill (more than 170 were hospitalized) and four young children died (P. Recer. "Experts call for irradiation of meat to protect against food-borne bacteria. " Associated Press. 7/12/94 [ 1994]). Several outbreaks since then have underscored the potential severity and multiple mechanisms for transmission of VTEC-associated diseases (M. Bielaszewska et al.. "Verotoxigenic (enterohaemorrhagic) Escherichia coli in infants and toddlers in Czechoslovakia," Infection 18: 352-356 [1990]: A. Caprioli et al.. "Hemolytic-uremic syndrome and Vero cytotoxin-producing Escherichia coli infection in Italy. "J. Infect. Dis.. 166: 184-158 [1992]; A. Caprioli. et al.. "Community-wide Outbreak of Hemolytic-Uremic Syndrome Associated with Non-0157 Verocytotoxin- Producing Escherichia coli." J. Infect. Dis.. 169: 208-21 1 [1994]: N. Cimolai. "Low frequency of high level Shiga-like toxin production in enteropathogenic Escherichia coli serogroups." Eur. J. Pediatr.. 151 : 147 [ 1992]: and R. Voelker.. "Panel calls £. coli screening inadequate." Escherichia coli O157:H7~Panel sponsored by the American Gastroenterological Association Foundation in July 1994. Medical News & Perspectives. J. Amer. Med. Assoc. 272: 501 [1994]). While O157:H7 is currently the predominant £. coli serotype associated with illness in North America, other serotypes (as shown in Table 1. and in particular O26:H l 1. O l 13 :H21. O91 :H21 and Ol 1 1 :NM) also produce verotoxins which appear to be important in the pathogenesis of gastrointestinal manifestations and the hemolvtic uremic syndrome (P. M. Griffin and R. V. Tauxe. "The epidemiology of infections caused by Escherichia coli O 157:H7. other enterohemorrhagic £. coli. and the associated hemolvtic uremic syndrome." Epidemiol. Rev.. 13: 60 [ 1990]: M. M. Levine. et ai. "Antibodies to Shiga holotoxin and to two synthetic peptides of the B subunit in sera of patients with Shigella dysenteήae 1 - _ - dysentery." J. Clin. Microbiol.. 30: 1636-1641 [1992]: and C. R. Dorn. et al.. "Properties of Vero cytotoxin producing Escherichia coli of human and animal origin belonging to serotypes other than O157:H7." Epidemiol. Infect., 103: 83-95 [1989]). Since organisms with these serotypes have been shown to cause illness in humans they may assume greater public health importance over time (P. M. Griffin and R. V. Tauxe. "The epidemiology of infections caused by Escherichia coli O157:H7. other enterohemorrhagic £. coli. and the associated hemolvtic uremic syndrome." Epidemiol. Rev., 13: 60 [1990]). Clinicians usually observe cases of hemoiytic uremic syndrome ("HUS") clustered in a geographic region. However, small outbreaks are likely to be missed because many laboratories do not routinely screen stool specimens for £ coli O157:H7. Many cases related to non-commercial food preparation also probably go unrecognized. Nonetheless. £. coli O157:H7 is responsible for a large number of cases, as more than 20.000 cases of £ coli O157:H7 infection are reported annually in the U.S.. with 400-500 deaths from HUS. However, these estimates were compiled when only 1 1 states mandated reporting of £ coli O157:H7. Twenty-nine states have recently made £ coli O157:H7 infection a reportable disease (R. Voelker. "Panel calls £. coli screening inadequate: Escherichia coli O157:H7; panel sponsored by the American Gastroenterological Association Foundation in July 1994. Medical News & Perspectives," J. Amer. Med. Assoc. 272: 501 [1994]). Indeed, the Centers for Disease Control recently added £. coli O157:H7 to their list of reportable diseases ("Public Health Threats." Science 267: 1427 [1995]). C. Nature of Verotoxin-Induced Disease Risk factors for HUS progression following infection with £. coli O157:H7 include age (very young or elderly), bloody diarrhea, leukocytosis. fever, large amounts of ingested pathogen, previous gastrectomy. and the use of antimicrobial agents (in particular. trimethoprim-sulfamethoxazole)(A. A. Harris et al.. "Results of a screening method used in a 12 month stool survey for Escherichia coli O157:H7." J. Infect. Dis.. 152: 775-777 [1985]: and M. A. Karmali. "Infection by Verocytotoxin-producing Escherichia coli." Clin. Microbiol. Rev.. 2: 15-38 [1989]). As indicated above. £. coli O157:H7 is associated with significant morbidity and mortality. The spectrum of illness associated with £. coli O157:H7 infection includes asymptomatic infection, mild uncomplicated diarrhea, hemorrhagic colitis. HUS. and TTP". Hemorrhagic colitis (or "ischemic colitis") is a distinct clinical syndrome characterized b> sudden onset of abdominal cramps — likened to the pain associated with labor or appendicitis — followed within 24 hours by watery diarrhea. One to two days later, the diarrhea turns grossly bloody in approximately 90% of patients and has been described as "all blood and no stool" (C. H. Pai et al., "Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7," Ann. Intern. Med.. 101 : 738-742 [1984]: and R. S. Remis et ai. "Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7." Ann. Intern. Med.. 101 : 738-742 [1984]). Vomiting may occur, but there is little or no fever. The time from ingestion to first loose stool ranges from 3-9 days (with a mean of 4 days) L. W. Riley et ai. "Hemorrhagic colitis associated with a rare Escherichia coli serotype." New Eng. J. Med.. 308: 681-685 [1983]; and D. Pudden et ai . "Hemorrhagic colitis in a nursing home." Ontario Can. Dis. Weekly Rpt.. 1 1 : 169-170 [1985]), and the duration of illness ranges generally from 2-9 days (with a mean of 4 days). HUS is a life-threatening blood disorder that appears w ithin 3-7 days following onset of diarrhea in 10-15% of patients. Those younger than 10 years and the elderly are at particular risk. Symptoms include renal glomerular damage, hemolvtic anemia (rupturing of erythrocytes as they pass through damaged renal glomeruli). thrombocytopenia and acute kidney failure. Approximately 15% of patients with HUS die or suffer chronic renal failure. Indeed, HUS is a leading cause of renal failure in childhood ( reviewed by M.A. Karmali. "Infection by Verocytotoxin-producing Escherichia coli." Clin. Microbiol. Rev.. 2: 15-38 [1989]). Currently, blood transfusion and dialysis are the only therapies for HUS. TTP shares similar histopathologic findings with HUS. but usually results in muitiorgan microvascular thrombosis. Neurological signs and fever are more prominent in TTP. compared with HUS. Generally occurring in adults. TTP is characterized by microangiopathic hemoiytic anemia, profound thrombocytopenia. fluctuating neurologic signs. fever and mild azotemia (H. C. Kwaan. "Clinicopathological features of thrombotic thrombocytopenic purpura." Semin. Hematol.. 24: 71-81 [1987]: and S. J. Machin. "Clinical annotation: Thrombotic thrombocytopenic purpura." Br. J. Hematol.. 56: 191 -197 [1984]). Patients often die from microthrombi in the brain. In one review of 271 cases, a rapidly progressive course was noted, with 75% of patients dying within 90 days (E.L. Amorosi and J.E. Ultmann. "Thrombotic thrombocytopenic purpura: Report of 16 cases and review of the literature." Med.. 45: 139-159 (1966). Other diseases associated with E. coli O157:H7 infection include hemorrhagic cystitis and balantitis (W. R. Grandsen et ai . "Hemorrhagic cystitis and balantitis associated with verotoxin-producing Escherichia coli O157:H7." Lancet ii: 150 [1985]). convulsions, sepsis with other organisms and anemia (P. C. Rowe et al. , "Hemoiytic anemia after childhood Escherichia coli O157:H7 infection: Are females at increased risk?" Epidemiol. Infect.. 106: 523-530 [1991]). D. Mechanism of Pathogenesis Verotoxins are strongly linked to E. coli O157:H7 pathogenesis. All clinical isolates of E. coli O157:H7 have been shown to produce one or both verotoxins (VT1 and VT2) (C. A. Bopp et ai. "Unusual Verotoxin-producing Escherichia coli associated with hemorrhagic colitis." J. Clin. Microbiol.. 25: 1486-1489 [1987]). Both of these toxins are cytotoxic to Vero (African green monkey kidney) and HeLa cells, and cause paralysis and death in mice (A. D. O ' Brien et ai. "Purification of Shigella dysenteriae 1 (Shiga) like toxin from Escherichia coli O157:H7 strain associated with hemorrhagic colitis." Lancet ii: 573 [1983]). These toxins are sometimes referred to in the literature as Shiga-like toxins I and II (SLT-I and SLT-II. respectively), due to their similarities with the toxins produced by Shigella. Indeed, much of our understanding of E. coli VTs is based on information accumulated on Shiga toxins. Shiga toxin, first described in 1903. has been recognized as one of the most potent bacterial toxins for eukaryotic cells (reviewed by M.A. Karmali. "Infection by Verocytotoxin-producing Escherichia coli " Clin. Microbiol. Rev.. 2: 15-38 [1989]). Hereinafter, the VT convention will be used; thus. VT1 and VT2 correspond to SLT-I and SLT-II. respectively. While the pathogenic mechanism of E. coli O157:H7 infection is incompletely und rstood, it is believed that ingested organisms adhere to and colonize the intestinal mucosa. where toxins are released which cause endothelial cell damage and bloody diarrhea. It is also postulated that hemorrhagic colitis progresses to HUS when verotoxins enter the bloodstream, damaging the endothelial cells of the microvasculature and triggering a cascade of events resulting in thrombus deposition in small vessels. These microthrombi occlude the microcapillaries of the kidneys (particularly in the glomeruli) and other organs, resulting in their failure (J. J. Byrnes and J. L. Moake. "TTP and HUS syndrome: Evolving concepts of pathogenesis and therapy." Clin. Hematol.. 15: 413-442 [1986]: and T. G. Cleary. "Cytotoxin- producing Escherichia coli and the hemoiytic uremic syndrome." Pediatr. Clin. North Am.. 35: 485-501 [ 1988]). Verotoxins entering the bloodstream may also result in direct kidne> cvtotoxicitv. VT1 is immunologically and structurally indistinguishable from Shiga toxin produced by Shigella dysenteriae (A. D. O'Brien et ai. "Purification of Shigella dysenreriae 1 (Shiga) like toxin from Escherichia coli O157:H7 strain associated with hemorrhagic colitis." Lancet ii: 573 [1983]). VT1 and VT2 holotoxins each consist of one A and five B subunits (A. Donohue-Rolfe et ai. "Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized PI glycoprotein and production of cross reactive monoclonal antibodies," Infect. Immun.. 57: 3888-3893 [1989]; and A. Donohue- Rolfe et ai. "Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies." J. Exp. Med.. 160: 1767-1781 [1984]). The toxic A subunit is enzymatically active, while the B subunit binds the holotoxin to the receptor on the target eukaryotic cell. Crystal structure analysis of Shiga holotoxin and VT1 B subunit pentamers have shown that the holotoxin assembles with the C-terminal end of the A subunit associating with, and inserting within, a pentamer of B chains (P. E. Stein et ai. "Crystal structure of the cell- binding B oligomer of verotoxin- 1 from £. coli." Nature 355: 748-750 [ 1992]: and M.E. Fraser et ai. "Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 A resolution." Struct. Biol.. 1 :59-64 [1994] ). This conformation is consistent with the observation that a C- terminally truncated Al subunit of VT1 is toxic (in a ribosomal inhibition assay), but cannot associate with B subunit pentamers (P. R. Austin et al. "Evidence that the A : fragment of Shiga-like toxin type I is required for holotoxin integrity." Infect. Immun.. 62: 1768 [1994]). The Verotoxin A Subunit. Examination of the crystal structure of Shiga holotoxin indicates that the N-terminus of its A subunit is both surface-exposed and functional!) important. Removal of amino acid interval 3-18 of the A subunit completely abolished toxicity (L. P. Perera et ai. "Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II." Infect. Immun.. 59: 829-835 [ 1991 ]) while removal of interval 25^ 4 retained toxicity but abolished its association with B subunit pentamers (J. E. Haddad et ai. "Minimum domain of the Shiga toxin A subunit required for enzymatic activity." J. Bacteriol.. 175: 4970-4978 [1993]). Deletion of the first 13 residues of the homologous ricin A subunit also abolished toxicity. while deletion of the first 9 residues did not (M. J. May. et ai. "Ribosome inactivation by ricin A chain: A sensitive method to assess the activity of wild-type and mutant polypeptides. " EMBO J.. 8: 301 -308 [ 1989]). The Verotoxin B Subunit. Studies of Shiga toxin B subunit suggest that neutralizing epitopes may also be present at both the N- and C-terminal regions of VT 1 and VT2 B subunits. Polyclonal antibodies raised against peptides from these regions (residues 5-18. 13-26. 7-26. 54-67 and 57-67) show partial neutralization of Shiga toxin (I. Harari and R. Arnon. "Carboxy-terminal peptides from the B subunit of Shiga toxin induce a local and parenteral protective effect." Mol. Immunol.. 27: 613-621 [1990]: and I. Harari et ai . "Synthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity." Infect. Immun., 56: 1618-1624 [1988]). Deletion of the last five amino acids of Shiga toxin B (M. P. Jackson et ai. "Functional Analysis of the Shiga toxin and Shiga-like toxin Type II variant binding subunits by using site-directed mutagenesis." J. Bacteriol.. 172: 653-658 [ 1990]). or four amino acids of VT2 B (L. P. Perera et ai . "Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II." Infect. Immun.. 59: 829-835 [ 1991 ]). eliminate toxin activity, while deletion of the last two amino acids of VT2 B subunit reduced cytotoxicity. In contrast, the addition of an 18 or 21 amino acid extension to the native C-terminus of the VT2 B subunit was presumably conformationally correct, as these proteins assembled cytotoxic holotoxin. Various approaches to express recombinant verotoxins have included individual or coordinate expression of A and B subunits from high-copy number plasmids and expression with fusion partners (J. E. Haddad et ai . "Minimum domain of the Shiga toxin A subunit required for enzymatic activity." J. Bacteriol.. 175: 4970-4978 : J. E. Haddad. and M. P. Jackson. "Identification of the Shiga toxin A-subunit residues required for holotoxin assembly." J. Bacteriol.. 175: 7652-7657 [1993]: M. P. Jackson et ai . "Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits." J. Bacteriol.. 1 72: 3346-3350 [ 1990]: C. J. Hovde et ai . "Evidence that glutamic acid 167 is an active-site residue of Shiga- like toxin I." Proc. Natl. Acad. Sci.. 85: 2568-2572 [ 1988]: R. L. Deresiewicz et ai . "The role of tyrosine-1 14 in the enzymatic activity of the Shiga-like toxin I A-chain." Mol. Gen. Genet.. 241 : 467-473 [ 1993]: T. M. Zoll an et ai . "Purification of Recombinant Shiga-like Toxin Type I A, Fragment from Escherichia coli." Protein Express. Purific. 5: 291 -295 [ 1994]; K. Ramotar. et ai . "Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron." Biochem J.. 272: 805-81 1 [ 1990]: S. B. Calderwood _?/ ai . "A system for production and rapid purification of large amounts of the Shiga toxin Shiga-like toxin I B subunit." Infect. Immun.. 58: 2977-2982 [ 1990]: D. W. K. Acheson. et ai . "Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or Iron-regulated promoter systems." Infect. Immun. 61 : 1098- 1 104 [ 1993]: M. P. Jackson el ai . "Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933," FEMS Microbiol. Lett.. 44: 109-1 14 [1987]; J. W. Newland et ai, "Cloning of genes for production of Escherichia coli Shiga-like toxin type II." Infect. Immun. 55: 2675-2680 [1987]; and F. Gunzer and H. Karch. "Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione 5-transferase and their potential for use in seroepidemiology.". J. Clin. Microbiol.. 31 : 2604-2610 [ 1993]: and D.W. Acheson et ai. "Expression and purification of Shiga-like toxin II B subunits." Inf. Immun.. 63:301 -308 [1995] ). In one case, bench top fermentation techniques yielded 22 mg/liter of soluble recombinant protein (D. W. K. Acheson. et ai. "Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or Iron- regulated promoter systems." Infect. Immun. 61 : 1098-1 104 [1993]). However, there have been no systematic approaches to identifying the appropriate spectrum of VT antigens, preserving immunogen and immunoabsorbant antigenicity and scaling-up. The receptor for VT1 and VT2 is a globotriaosyl ceramide containing a galactose α-( l -4)- galactose-β-( l -4) glucose ceramide (Gb3) (C. A. Lingwood et ai. "Glycolipid binding of natural and recombinant Escherichia coli produced verotoxin in vitro." J. Biol. Chem.. 262: 1779-1785 [1987]: and T. Wadell et ai . "Globotriaosyl ceramide is specifically recognized by the Escherichia coli verocytotoxin 2." Biochem. Biophys. Res. Commun.. 152: 674-679 [1987]). Gb3 is abundant in the cortex of the human kidney and is present in primary human endothelial cell cultures. Hence, the identification of Gb3 as the functional receptor for VT1 and VT2 is consistent with their role in HUS pathogenesis. in which endothelial cells of the renal vasculature are the principal site of damage. Therefore, toxin- mediated pathogenesis may follow a sequence of B subunit binding to Gb3 receptors on kidney cells, toxin internalization. enzymatic reduction of the A subunit to an A 1 fragment. binding of the Al subunit to the 60S ribosomal subunit. inhibition of protein synthesis and cell death (A. D. O ' Brien et ai. "Shiga and Shiga-like toxins. Microbial Rev.. 51 : 206-220 [1987]). The role of verotoxins in the pathogenesis of £. coli O157:H7 infections has been further studied in animal models. Infection or toxin challenge of laboratory animals do not produce all the pathologies and symptoms of hemorrhagic colitis. HUS. and TTP which occur in humans. Glomerular damage is noticeably absent. Nonetheless, experiments using animal models implicate verotoxins as the direct cause of hemorrhagic colitis, microvascular damage leading to the failure of kidneys and other organs and CNS neuropathies. For example. Barrett, et al. delivered VT2 into the peritoneal cavity of rabbits using mini-osmotic pumps (J. J. Barrett et ai . "Continuous peritoneal infusion of shiga-like toxin II (SLTII) as a model for SLT II-induced diseases." J. Infect. Dis.. 159: 774-777 [1989]). In three days, most animals receiving the toxin developed diarrhea, w ith intestinal lesions resembling those seen in humans with hemorrhagic colitis. Although there was some evidence of renal dysfunction, none of the rabbits developed HUS. Beery, et al. showed that VT2, when administered intraperitoneally or intravenously to adult mice, produces lesions of the kidneys and colon (J. T. Beery et ai. "Cytotoxic activity of Escherichia coli O157:H7 culture filtrate on the mouse colon and kidney." Curr. Microbiol.. 1 1 : 335-342 [ 1984]). Histologic lesions in the kidney included accumulation of numerous exfoliated collecting tubules and marked intracellular vacuolation of proximal convoluted tubular cells. Sjδgren et. ai studied the pathogenesis of an entero-adherent strain of £. coli ( RDEC- 1 ) lysogenized with a VTl -containing bacteriophage (VTl -producing RDEC- 1 ) (R. Sjδgren et ai . "Role of Shiga-like toxin I in bacterial enteritis: comparison between isogenic Escherichia coli strains induced in rabbits." Gastroenterol.. 106: 306-31 7 [ 1994]). In this study, rabbits were challenged with RDEC-1 or VTl -producing RDEC- 1 and studied for onset of disease. The VTl -producing variant induced a severe, non-invasive, entero-adherent infection in rabbits which was characterized by serious histological lesions with vascular changes, edema and severe epithelial inflammation. Importantly, vascular changes consistent with endothelial damage were seen in infected animals that was similar to intestinal microvascular changes in humans with £. coli O157:H7 infection. Based on these observations, they concluded that VTl is an important virulence factor in enterohemorrhagic £. coli O157:H7 infection. Fuji et. al. described a model in which mice were treated for three days with streptomycin followed by a simultaneous challenge of £. coli O157:H7 orally, and mitomycin intraperitoneally (J. Fuji et ai. "Direct evidence of neuron impairment by oral infection with Verotoxin-producing Escherichia coli O157:H7 in mitomycin-treated mice." Infect. Immun.. 62: 3447-34453 [1994]). All of the animals died within four days. Immunoelectron- microscopy strongly suggested that death was due to the toxic effects of VT2v (a structural variant of VT2), on both the endothelial cells and neurons in the central nervous system which resulted in fatal acute encephalopathy. Wadolkowski et al. studied colonization of £ coli O157:H7 in mice. Mice were treated with streptomycin and fed 10 lϋ £ coli O157:H7 (E. A. Wadolkowski et ai . "Mouse model for colonization and disease caused by enterohemorrhagic Escherichia toll 0157 H7." Infect Immun . 58 2438-2445 [1990]. and E A Wadolkowski et al "Acute renal tubular necrosis and death of mice orally infected with Escherichia toll strains that produce Shiga- like toxin Type II." Infect Immun . 58 3959-3965 [1990]) All of the mice died due to severe, disseminated, acute necrosis of proximal convoluted tubules In mouse models, glomerular damage was not observed, but toxic acute renal tubular necrosis was observed which is characteristic of some HUS patients The failure ol mice to show glomerular damage is thought to be due to the absence of a functional globotriaosyl ceramide receptor specific for verotoxins in the glomeruli of the kidneys Administration of VT2 subunit- specific monoclonal antibodies prior to infection prevented all pathology and death E. Current Therapeutic Approaches £ coli 0157 H7 disease is not adequately controlled by current therapy Patient treatment is tailored to manage fluid and electrolyte disturbances, anemia renal failure and hypertension Although £ coli 0157 H7 is susceptible to common antibiotics, the role of antibiotics in the treatment of infection has questionable merit In both retrospectiv e and prospectiv e studies, prophylaxis or treatment with antibiotics such as tπmethopπm- sulfamethoxazole. there was either no benefit or an increased risk of developing HUS (T N Bokete et al . "Shiga-like toxin producing Escherichia coli in Seattle children a prospectiv e study." Gastroenterol . 105 1724-1731 [1993], A T Pavia ei al . "Hemolvtic uremic- syndrome duπng an outbreak of Escherichia coli 0157 H7 infections in institutions for mentally retarded persons clinical and epidemiologic observ ations " J Pedatr 1 16 544-551 [ 1990] F Proul \ et al "Randomized, controlled trial of antibiotic therapy tor Escherichia coli 0157 H7 enteritis ' J Pediatr 121 299-303 [ 1992]. and A L Carter et al " A severe outbreak of Escherichia coli 0157 H7-assocιated hemorrhagic colitis in a nursing home New Eng J Med . 317 1496-1500 [ 1987]) The mechanisms by which antibiotics increase the risk of infection or related complications might inv olv e enhancement of toxin production, release of toxins from killed organisms, or alteration of normal competing intestinal flora allowing for pathogen overgrowth (M A Karmali "Infection by Verocytotoxin-producing Escherichia coli " Clin Microbiol Rev . 2 15-38 [ 1989]) A further concern in the use of antibiotics is the potential acquisition of antimicrobial resistance by £ coli 0157 H7 (C R Dorn "Rev iew of foodborne outbreak of Escherichia coli 0157 H7 infection in the western United States ' J ΛVM Λ 203 1583- 1587 [ 1993] ) In addition, by the time symptoms are serious enough to attract medical attention, it is likely that verotoxins are already entering the systemic circulation or will do so shortly thereafter. Although antimicrobials may help to prevent pathology resulting from the action of toxin on the bowel lumen. However, by the time symptoms of HUS have developed, the patient has ceased shedding organisms. Thus, antimicrobial treatment during HUS disease is of less value, and often contraindicated. due to the increased risk of complications associated with administration of antimicrobials to patients susceptible to development of HUS. Importantly, there is currently no antitoxin commercially available for use in treating affected patients. What is needed is a means to block the progression of disease, without the complications associated with antimicrobial treatment. DESCRIPTION OF THE DRAWINGS Figure 1 is an SDS-PAGE of rVTl and rVT2. Figure 2 shows HPLC results for rVTl and rVT2. Figure 3 shows rVTl and rVT2 toxicity in Vero cell culture. Figure 4 shows El A reactivity of rVTl and rVT2 antibodies to rVTl . Figure 5 shows EIA reactivity of rVTl and rVT2 Antibodies to rVT2. Figure 6 shows Western Blot reactivity of rVTl and rVT2 antibodies to rVT ' s: Panel 6A contains preimmune IgY: Panel 6B contains rVTl IgY; and Panel 6C contains rVT2 IgY. Figure 7 shows neutralization of rVTl cytotoxicit in Vero cells. Figure 8 shows neutralization of rVT2 cytotoxicity in Vero cells. Figure 9 shows renal sections from £ coli O157:H7-infected mice treated with IgY Panel 9A shows a representative kidne section from a mouse treated with preimmune IgY; Panel 9B shows a representative kidney sections from a mouse treated with rVT and Panel 9C shows a representative kidne section from a mouse treated with rVT2 IgY. Figure 10 shows the fusion constructs of VT components and affinity tags. DEFINITIONS To facilitate understanding of the invention, a number of terms are defined below As used herein, the term "neutralizing" is used in reference to antitoxins, particularly antitoxins comprising antibodies, which have the ability to prev ent the pathological actions of the toxin against which the antitoxin is directed As used herein, the term "overproducing" is used in leteience to the production of toxin polypeptides in a host cell, and indicates that the host cell is producing more of the toxin by virtue of the introduction of nucleic acid sequences encoding the toxin polypeptide than would be expressed by the host cell absent the introduction of these nucleic acid sequences To allow ease of purification of toxin polypeptides produced in a host cell it is preferred that the host cell express or overproduce the toxin poly peptide at a level greater than 1 mg/liter of host cell culture As used herein, the term "fusion protein" refers to a chimeπc protein containing the protein of interest (i e an £ coli verotoxin and/or fragments thereof) |oιned to an exogenous protein fragment (the fusion partner which consists of a non-to\ιn protein) The fusion partner may enhance solubility of the £ coli protein as expressed in a host cell, mav provide an "affinity tag" to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both If desired, the fusion protein mav be removed from the protein of interest (; e . toxin protein or fragments thereof) prior to immunization by a variety of enzv matic or chemical means known to the art As used herein, the term "affinity tag ' refers to such stiuctures as a 'polv -histidine tract or poly-histidine tag." or any other structure or compound which facilitates the purification of a recombinant fusion protein from a host cell, host cell culture supernatant, or both As used herein, the term "flag tag" refers to short polv peptide marker sequence useful for recombinant protein identification and purification As used herein, the terms "poly-histidine tract" and "polv -histidine tag." when used in reference to a fusion protein refers to the presence of two to ten histidine residues at either the amino- or carboxy -terminus of a protein of interest A poly -histidine tract of six to ten residues is preferred The poly-histidine tract is also defined functionally as being a number of consecutive histidine residues added to the protein of interest which allows the affinity purification of the resulting fusion protein on a nickel-chelate column As used herein, the term "chimeπc protein" refers to two or more coding sequences obtained from different genes, that have been cloned together and that, after translation, act as a single polypeptide sequence. Chimeric proteins are also referred to as "hy brid proteins." As used herein, the term "chimeric protein" refers to coding sequences that are obtained from different species of organisms, as well as coding sequences that are obtained from the same species of organisms. As used herein, the term "protein of interest" refers to the protein whose expression is desired within the fusion protein. In a fusion protein, the protein of interest will be joined or fused with another protein or protein domain, the fusion partner, to allow for enhanced stability of the protein of interest and/or ease of purification of the fusion protein. As used herein, the term "maltose binding protein" and "MBP" refers to the maltose binding protein of £. coli. A portion of the maltose binding protein may be added to a protein of interest to generate a fusion protein: a portion of the maltose binding protein may merely enhance the solubility of the resulting fusion protein when expressed in a bacterial host. On the other hand, a portion of the maltose binding protein may allow affinity purification of the fusion protein on an amylose resin. As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, antitoxins are purified by remov al of contaminating non-immunoglobulin proteins; they are also purified by the removal of substantially all immunoglobulin that does not bind toxin. The remov al of non- immunoglobulin proteins and/or the removal of immunoglobulins that do not bind toxin results in an increase in the percent of toxin-reactive immunoglobulins in the sample. In another example, recombinant toxin polypeptides are expressed in bacterial host cells and the toxin polypeptides are purified by the removal of host cell proteins: the percent of reco nbinant toxin polypeptides is thereby increased in the sample. The term "recombinant DNA molecule" as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule which is expressed from a recombinant DNA molecule. The term "native protein" as used herein refers to a protein which is isolated from a natural source as opposed to the production of a protein by recombinant means. As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. As used herein "soluble" when in reference to a protein produced by recombinant DNA technology in a host cell, is a protein which exists in solution in the cytoplasm of the host cell; if the protein contains a signal sequence, the soluble protein is exported to the periplasmic space in bacterial hosts and is secreted into the culture medium of eukaryotic cells capable of secretion or by bacterial hosts possessing the appropriate genes. In contrast, an insoluble protein is one which exists in denatured form inside cytoplasmic granules (called an inclusion bodies) in the host cell. High level expression (i.e.. greater than 1 mg recombinant protein/liter of bacterial culture) of recombinant proteins often results in the expressed protein being found in inclusion bodies in the bacterial host cells. A soluble protein is a protein which is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm. A distinction is drawn between a soluble protein (i.e.. a protein which when expressed in a host cell is produced in a soluble form) and a "solubilized" protein. An insoluble recombinant protein found inside an inclusion body may be solubilized (/ e.. rendered into a soluble form) by treating purified inclusion bodies with denaturants such as guanidine hydrochloride. urea or sodium dodecyl sulfate (SDS). These denaturants must then be removed from the solubilized protein preparation to allow the recovered protein to renature (refold). Not all proteins will refold into an active conformation after solubilization in a denaturant and remov al of the denaturant. Many proteins precipitate upon removal of the denaturant. SDS may be used to solubilize inclusion bodies and will maintain the proteins in solution at low concentration. However, dialysis will not always remove all of the SDS ( SDS can form micelles which do not dialyze out): therefore. SDS-solubilized inclusion body protein is soluble but not refolded. As used herein, the term "reporter reagent" or "reporter molecule" is used in reference to compounds which are capable of detecting the presence of antibody bound to antigen. For example, a reporter reagent may be a colorimetric substance which is attached to an enzymatic substrate. Upon binding of antibody and antigen, the enzyme acts on its substrate and causes the production of a color. Other reporter reagents include, but are not limited to fluorogenic and radioactive compounds or molecules. As used herein the term "signal" is used in reference to the production of a sign that a reaction has occurred, for example, binding of antibody to antigen. It is contemplated that signals in the form of radioactivity, fluorogenic reactions, and enzymatic reactions will be used with the present invention. The signal may be assessed quantitativ ely as well as qualitatively. As used herein, the term "therapeutic amount" refers to that amount of antitoxin required to neutralize the pathologic effects of £. coli toxin in a subject. As used herein, the term "acute intoxication" is used in reference to cases of E. coli infection in which the patient is currently suffering from the effects of toxin (e.g., E. coli verotoxins or enterotoxins). Signs and symptoms of intoxication with the toxin may be immediately apparent. Or. the determination of intoxication ma require additional testing, such as detection of toxin present in the patient ' s fecal material. As used herein, the term "at risk" is used in references to individuals who have been exposed to £. coli and may suffer the symptoms associated with infection or disease with these organisms, especially due to the effects of verotoxins. SUMMARY OF THE INVENTION The present invention relates to antitoxin therapy for humans and other animals. Antitoxins which neutralize the pathologic effects of £ coli toxins are generated by immunization of avian hosts with recombinant toxin fragments. In one embodiment, the present invention contemplates a method of treatment administering at least one antitoxin directed against at least a portion of an Escherichia coli verotoxin in an aqueous solution in therapeutic amount that is administrable to an intoxicated subject. It is contemplated that the intoxicated subject will be either an adult or a child. In a preferred embodiment, the £. coli verotoxin is recombinant. In one embodiment, the antitoxin is an avian antitoxin. In an alternative embodiment, the recombinant £. coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the Escherichia cυli verotoxin VTl sequence. In one embodiment of the E. coli fusion protein, the fusion protein comprises a non-verotoxin protein sequence and a portion of the Escherichia coli v erotoxin VT2 sequence. Various routes of administration, are contemplated for providing the £ coli antitoxin(s) to an affected individual, including but not limited to. parenteral as well as oral routes of administration. In a particularly preferred embodiment, the route of administration is parenteral. The present invention also includes the embodiment of a method of prophylactic treatment in which an antitoxin directed against at least one £ coli verotoxin in an aqueous solution in therapeutic amount that is parenterallv administrable. and is administered to at least one subject at risk of diarrheal disease. It one embodiment, the antitoxin is parenterallv administered. In one embodiment, the subject is at risk of developing extra-intestinal complications of £. coli infections, including but not limited to. hemoiytic uremic syndrome, thrombotic thrombocytopenic purpura. etc. The present invention also includes the embodiment of a composition which comprises neutralizing antitoxin directed against at least one £. coli verotoxin in an aqueous solution in therapeutic amounts. In one particularly preferred embodiment, the £ coli verotoxin is a recombinant toxin. In an alternative embodiment, the recombinant £ coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the £ coli verotoxin VTl sequence. In another embodiment, the recombinant £. coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the £ coli verotoxin VT2 sequence. In yet another embodiment, the composition of the antitoxin is directed against a poπion of at least one Escherichia coli verotoxin. In one embodiment, the portion of Escherichia coli is selected from the group consisting of subunit A and subunit B of VTl . In an alternative embodiment, the portion f Escherichia coli is selected from the group consisting of subunit A and subunit B of VT2. Indeed, the invention contemplates an antitoxin that is directed against a portion of at least one Escherichia coli verotoxin. In one embodiment, the antitoxin is an avian antitoxin. The present invention also comprises a method of treatment of enteric bacterial infections comprising administering an avian antitoxin directed against at least one verotoxin produced by £. coli in an aqueous solution in therapeutic amount, to at least one infected subject. In one preferred embodiment, the avian antitoxin is administered parenterally. In another embodiment, the £. coli is selected from the group consisting of Escherichia coli serotypes O157:H7. Ol :NM: O2:H5: O2:H7: O4:NM: O4:H10: O5:NM: O5:H16: O6:Hl: O18:NM: O18:H7; O25:NM: O26:NM: O26:Hl l : O26:H32; O38:H21 ; O39:H4: O45:H2; O50:H7: O55:H7: O55:H10: O82:H8: O84:H2: O91 :NM: O91 :H21 ; O103:H2: Ol l l :NM: Ol l l :H8: O1 1 1.H30: Ol l l :H34: O1 13:H7: OI 13:H21 : O1 14:H48: O1 15:H10: O1 17:H4: O1 18:H12: O1 18:H30: O121 :NM: O121 :H 19: O125:NM: O125:H8: O126.NM: O126:H8: O128.NM: O128:H2: O128:H8: O128:H12: O128:H25: O145.NM: O125:H25: O146:H21 : O153.H25: O157:NM: O163:H19: O165:NM: 0165: 19; and O165:H25. In one embodiment, the antitoxin comprises antitoxin directed against at least one Escherichia coli verotoxin. In another embodiment, the antitoxin is cross-reacts with at least one Escherichia coli verotoxin. In yet another embodiment, the antitoxin is reactive against toxins produced by members of the genus Shigella. including S. dysenteriae. The present invention also contemplates uses for the toxin fragments in vaccines and diagnostic assays. The fragments may be used separately as purified, soluble antigens or. alternatively, in mixtures or "cocktails." The present invention thus comprises a method for detecting Escherichia coli verotoxin in a sample in which a sample.an antitoxin raised against Escherichia coli verotoxin. and a reporter reagent capable of binding the antitoxin are provided. The antitoxin is added to the sample, so that the antitoxin binds to the £ coli verotoxin in the sample. In one embodiment, the antitoxin is an avian antitoxin. In an alternative embodiment, the method further comprises the steps of washing unbound antitoxin from the sample, adding at least one reporter reagent to the sample, so that said reporter reagent binds to any antitoxin that is bound, washing the unbound reporter reagent from the sample and detecting the reporter reagent bound to the antitoxin bound to the Escherichia coli verotoxin. so that the verotoxin is detected. In one embodiment, the detecting is accomplished through any means, such as enzyme immunoassay. radioimmunoassay. fluorescence immunoassay, flocculation. particle agglutination, and in suit chromogenic assay. In one preferred embodiment, the sample is a biological sample. In an alternative preferred embodiment, the sample is an environmental sample. DESCRIPTION OF THE INVENTION The present invention contemplates treating humans and other animals intoxicated with at h ast one bacterial toxin. It is contemplated that administration of antitoxin will be used to treat patients effected by or at risk of symptoms due to the action of bacterial toxins. It is also contemplated that the antitoxin will be used in a diagnostic assay to detect the presence of toxins in samples. The organisms, toxins and individual steps of the present invention are described separately below. I. Antibodies Directed Against E. coli and Associated Toxins A preferred embodiment of the method of the present invention is directed toward obtaining antibodies against various £ coli serotypes. their toxins, enzymes or other metabolic by-products, cell wall components, or synthetic or recombinant versions of any of these compounds. It is contemplated that these antibodies will be produced by immunization of humans or other animals. It is not intended that the present invention be limited to any particular toxin or any species of organism. In one embodiment, toxins from all £ coli serotypes are contemplated as immunogens. Examples of these toxins include the verotoxins VTl and VT2. It is not intended that antibodies produced against one toxin will only be used against that toxin. It is contemplated that antibodies directed against one toxin may be used as an effective therapeutic against one or more toxin(s) produced by other £. coli serotypes. or other toxin producing organisms (e.g.. Shigella. Bacillus cereus. Staphylococcus aureus. Streptococcus mutans. Acinetobacter calcoaceticus, Pseudomonas aeruginosa. other Pseudomonas species. Vibrio species. Clostridium species, etc. ). It is further contemplated that antibodies directed against the portion of the toxin which binds to mammalian membranes can also be used against other organisms. It is contemplated that these membrane binding domains are produced synthetically and used as immunogens. II. Obtaining Antibodies In Non-Mammals A preferred embodiment of the method of the present invention for obtaining antibodies involves immunization. However, it is also contemplated that antibodies may be obtained from non-mammals without immunization. In the case where no immunization is contemplated, the present invention may use non-mammals with preexisting antibodies to toxins as well as non-mammals that hav e antibodies to whole organisms by virtue of reactions with the administered antigen. An example of the latter involves immunization with synthetic peptides or recombinant proteins sharing epitopes with whole organism components. In a preferred embodiment, the method of the present invention contemplates immunizing non-mammals with bacterial toxin(s). It is not intended that the present invention be limited to any paπicular toxin. In one embodiment, toxins from all £. coli serotypes are contemplated as immunogens. A paπicularly preferred embodiment involves the use of bacterial toxin protein or fragments of toxin proteins produced by molecular biological means (i.e.. recombinant toxin proteins). In a preferred embodiment, the immunogen comprises recombinant VTl and/or VT2. When immunization is used, the preferred non-mammal is from the class Aves. All birds are contemplated [ e.g.. duck, ostrich, emu. turkey, etc.). A preferred bird is a chicken, lmpoπantly. chicken antibody does not fix mammalian complement (See H.N. Benson et ai . J. Immunol. 87:616 [ 1961]). Thus, chicken antibody will normally not cause a complement- dependent reaction (A. A. Benedict and K. Yamaga. "Immunoglobulins and Antibody Production in Avian Species, " in Comparative Immunology (J.J. Marchaloni. ed.), pp. 335- 375, Blackwell. Oxford [1966]). Thus, the preferred antitoxins of the present invention will not exhibit complement-related side effects observed with antitoxins presently known. When birds are used, it is contemplated that the antibody will be obtained from either the bird serum or the egg. A preferred embodiment involves collection of the antibody from the egg. Laying hens transpoπ immunoglobulin to the egg yolk ("IgY") in concentrations equal to or exceeding that found in serum (See R. Patterson el ai . J. Immunol. 89:272 ( 1962): and S.B. Carroll and B.D. Stollar. J. Biol. Chem. 258:24 [ 1983]). In addition, the large volume of egg yolk produced vastly exceeds the volume of serum that can be safely obtained from the bird over any given time period. Finally, the antibody from eggs is more pure and more homogeneous: there is far less non-immunoglobulin protein ( as compared to serum) and only one class of immunoglobulin is transported to the yolk. When considering immunization with toxins, one may consider modification of the toxins to reduce the toxicity. In this regard, it is not intended that the present invention be limited by immunization with modified toxin. Unmodified ("native") toxin is also contemplated as an immunogen. It is also not intended that the present invention be limited by the type of modification — if modification is used. The present invention contemplates all types of toxin modification, including chemical and heat treatment of the toxin. In one embodiment, glutaraldehyde treatment of the toxin is contemplated. In an alternative embodiment, formaldehyde treatment of the toxin is contemplated. It is not intended that the present invention be limited to a paπicular mode of immunization: the present invention contemplates all modes of immunization, including subcutaneous, intramuscular, intraperitoneal. and intravenous or intravascular injection, as well as per os administration of immunogen. The present invention fuπher contemplates immunization with or without adjuvant. As used herein, the term "adjuvant" is defined as a substance known to increase the immune response to other antigens when administered with other antigens. If adjuvant is used, it is not intended that the present invention be limited to any paπicular type of adjuvant - or that the same adiuvant. once used, be used all the time. \\ Tiile the present invention contemplates all types of adjuvant, whether used separately or in combinations, the preferred use of adjuvant is the use of Complete Freund ' s Adjuvant followed sometime later with Incomplete Freund ' s Adjuvant. The invention also contemplates the use of fowl adjuvant commercially- available from RIBL as well as Quil A adjuvant commercially av ailable from Accurate Chemical and Scientific Corporation, and Gerbu adjuvant also commercially available (GmDP; C.C. Biotech Corp.). When immunization is used, the present invention contemplates a wide variety of immunization schedules. In one embodiment, a chicken is administered toxin(s) on day zero and subsequently receives toxin(s) in intervals thereafter. It is not intended that the present invention be limited by the paπicular intervals or doses. Similarly, it is not intended that the present invention be limited to any paπicular schedule for collecting antibody. The preferred collection time is sometime after day 35. Where birds are used and collection of antibody is performed by collecting eggs, the eggs may be stored prior to processing for antibody. It is preferred that eggs be stored at 4°C for less than one year. It is contemplated that chicken antibody produced in this manner can be buffer- extracted and used analytically. While unpurified. this preparation can serve as a reference for activity of the antibody prior to fuπher manipulations (e.g.. immunoaffinity purification). III. Increasing The Effectiveness Of Antibodies When purification is used, the present invention contemplates purifying to increase the effectiveness of both non-mammalian antitoxins and mammalian antitoxins. Specifically, the present invention contemplates increasing the percent of toxin-reactive immunoglobulin. The preferred purification approach for avian antibody is polyethylene glycol (PEG) separation. The present invention contemplates that avian antibody be initially purified using simple, inexpensive procedures. In one embodiment, chicken antibody from eggs is purified by extraction and precipitation with PEG. PEG purification exploits the differential solubility of lipids (which are abundant in egg yolks) and yolk proteins in high concentrations of PEG 8000 (Poison et ai . Immunol. Comm. 9:495 [ 1980]). The technique is rapid, simple, and relatively inexpensive and yields an immunoglobulin fraction that is significantly more pure. in terms of contaminating non-immunoglobulin proteins than the comparable ammonium sulfate fractions of mammalian sera and horse antibodies. The majority of the PEG is remov ed from the precipitated chicken immunoglobulin by treatment with ethanol. Indeed. PEG-purified antibody is sufficiently pure that the present invention contemplates the use of PEG-purifϊed antitoxins in the passive immunization of intoxicated humans and animals. IV. Treatment The present invention contemplates antitoxin therapy for humans and other animals intoxicated by bacterial toxins. A preferred method of treatment is by parenteral administration of antitoxin. A. Dosage Of Antitoxin It was noted by way of background that a balance must be struck when administering currently available antitoxin which is usually produced in large animals such as horses: sufficient antitoxin must be administered to neutralize the toxin, but not so much antitoxin as to increase the risk of untoward side effects. These side effects are caused by : i) patient sensitivity to foreign (e.g. horse) proteins; ii) anaphylactic or immunogenic properties of non- immunoglobulin proteins; iii) the complement fixing properties of mammalian antibodies: and/or iv) the overall burden of foreign protein administered. It is extremely * difficult to strike this balance when, as noted above, the degree of intoxication (and hence the level of antitoxin therapy needed) can only be approximated. The present invention contemplates significantly reducing side effects so that this balance is more easily achieved. Treatment according to the present invention contemplates reducing side effects by using PEG-purified antitoxin from birds. In one embodiment, the treatment of the present invention contemplates the use of PEG-purified antitoxin from birds. The use of yolk-derived. PEG-purified antibody as antitoxin allows for the administration of: 1 ) non (mammalian (-complement-fixing, avian antibody: 2) a less heterogeneous mixture of non-immunoglobulin proteins: and 3) less total protein to deliver the equivalent weight of active antibody present in currently available antitoxins. The non-mammalian source of the antitoxin makes it useful for treating patients who are sensitive to horse or other mammalian sera. As is true in cases of botulism, the degree of an individual ' s exposure to £. coli toxin and the prognosis are often difficult to assess, and depend upon a number of factors (e.g.. the quantity of contaminated food ingested, the toxigenicity and serotype of £. coli strain ingested, etc.). Thus, the clinical presentation of a patient is usually a more important consideration than a quantitative diagnostic test, for determination of dosage in antitoxin 11 administration. Indeed, for many toxin-associated diseases (e.g.. botulism, tetanus, diphtheria, etc.). there is no rapid, quantitative test to detect the presence of the toxin or organism. Rather, these toxin-associated diseases are medical emergencies which mandate immediate treatment. Confirmation of the etiologic agent must not delay the institution of therapy, as the condition of an affected patient may rapidly deteriorate. In addition to the initial treatment with antitoxin, subsequent doses may be indicated, as the patient ' s disease progresses. The dosage and timing of these subsequent doses is dependent upon the signs and symptoms of disease in each individual patient. It is contemplated that the administration of antitoxin to an affected individual would involve an initial injection of an approximately 10 ml dose of immune globulin (with less than approximately 1 gram of total protein). In one preferred embodiment, it is contemplated that at least 50% of the initial injection comprises immune globulin. It is also contemplated that more purified immune globulin be used for treatment, wherein approximately 90% of the initial injection comprises immune globulin. When more purified immune globulin is used. it is contemplated that the total protein will be less than approximately 100 milligrams. It is also contemplated that additional doses be given, depending upon the signs and symptoms associated with £. coli verotoxin disease progression. B. Deliver}* Of Antitoxin Although it is not intended to limit the route of delivery, the present invention contemplates a method for antitoxin treatment of bacterial intoxication in which delivery of antitoxin is parenteral or oral. In one embodiment, antitoxin is parenterallv administered to a subject in an aqueous solution. It is not intended that the parenteral administration be limited to a particular route. Indeed, it is contemplated that all routes of parenteral administration will be used. In one embodiment, parenteral administration is accomplished via intramuscular injection. In an alternative embodiment, parenteral administration is accomplished via intrav enous injection. In another embodiment, antitoxin is delivered in a solid form (e.g.. tablets). In an alternative embodiment antitoxin is delivered in an aqueous solution. When an aqueous solution is used, the solution has sufficient ionic strength to solubilize antibody protein, yet is made palatable for oral administration. The delivery solution may also be buffered (e.g.. carbonate buffer. pH 9.5) which can neutralize stomach acids and stabilize the antibodies when the antibodies are administered orallv. In one embodiment the deliver, solution is an aqueous solution. In another embodiment the delivery solution is a nutritional formula. Preferably, the delivery solution is infant or a dietary supplement formula (e.g., Similac®, Ensured, and Enfamil®). Yet another embodiment contemplates the delivery of lyophilized antibody encapsulated or microencapsulated inside acid-resistant compounds. Methods of applying enteric coatings to pharmaceutical compounds are well known to the art (companies specializing in the coating of pharmaceutical compounds are available: for example. The Coating Place [Verona. WI] and AAI [Wilmington. NC]). Enteric coatings which are resistant to gastric fluid and whose release (i.e.. dissolution of the coating to release the pharmaceutical compound) is pH dependent are commercially available (for example, the polymethacrylates Eudragit® L and Eudragit® S [Rohm Tech Inc.. Maiden. MA]). Eudragit® S is soluble in intestinal fluid from pH 7.0; this coating can be used to microencapsulate lyophilized antitoxin antibodies and the particles are suspended in a solution having a pH above or below pH 7.0 for oral administration. The micropaπicles will remain intact and undissolved until they reached the intestines where the intestinal pH would cause them to dissolve thereby releasing the antitoxin. The invention contemplates a method of treatment which can be administered for treatment of acute intoxication. In one embodiment, antitoxin is administered orally in either a delivery solution or in tablet form, in therapeutic dosage, to a subject intoxicated by the bacterial toxin which served as immunogen for the antitoxin. In another embodiment of treatment of acute intoxication, a therapeutic dosage of the antitoxin in a delivery solution, is parenterallv administered. The invention also contemplates a method of treatment which can be administered prophylactically. In one embodiment, antitoxin is administered orally, in a delivery solution, in therapeutic dosage, to a subject, to prevent intoxication of the subject by the bacterial toxin which served as immunogen for the production of antitoxin. In another embodiment. antitoxin is administered orally in solid form such as tablets or as microencapsulated particles. Microencapsulation of lyophilized antibody using compounds such as Eudragit® (Rohm GmbH) or polyethylene glycol. which dissolve at a wide range of pH units, allows the oral administration of solid antitoxin in a liquid form (i.e.. a suspension) to recipients unable to tolerate administration of tablets (e.g., children or patients on feeding tubes). In one preferred embodiment the subject is a child. In another embodiment, antibody raised against whole bacterial organism is administered orally to a subject, in a delivery solution, in therapeutic dosage. In yet another preferred embodiment of prophylactic treatment, a therapeutic dosage of the antitoxin in a delivery solution, is parenterallv administered V. Multivalent Vaccines Against E. coli Strains The invention contemplates the generation of multivalent v accines for the protection of an organism (paπicularly humans) against several £ coli strains Of paπicular interest is a vaccine which stimulates the production of a humoral immune response to £ coli 0157 H7. 026 HI 1. Ol 13 H21. O91 -H21, and Ol 1 1 NM. in humans The antigens comprising the vaccine preparation may be native or recombinantly produced proteins from the £ coli serotypes listed above When native toxin proteins are used as immunogens they are generally modified to reduce the toxicity It is contemplated that glutaraldehyde-modified toxin proteins will be used In an alternative embodiment, is formaldeh de-modified toxin proteins will be used The invention contemplates that recombinant £ coli v erotoxin proteins be used in conjunction with either native toxins or toxoids from other organisms as antigens in a multivalent vaccine preparation It is also contemplated that recombinant £ coli toxin proteins be used the multivalent vaccine preparation VI. Detection Of Toxin The invention contemplates detecting bacterial toxin in a sample The term "sample" in the present specification and claims is used in its broadest sense On the one hand it is meant to include a specimen or culture (e g microbiological cultures) On the other hand it is meant to include both biological and environmental samples Biological samples may be animal, including human, fluid solid ( . stool) or tissue. as well as liquid and solid food and feed products and ingredients such as dairy items egetables, meat and meat by-products, and waste Biological samples mav be obtained from all of the various families of common domestic animals, including but not limited, to bov ines (e g cattle), ovines (e g sheep), capπnes (e g goats) porcines (c ,_: swine) equ es g horses), canines (e g dogs), lagamorphs (e g rabbits), and felines (e g cats), etc It is also intended that samples mav be obtained from feral or wild animals including, but not limited to. such animals as ungulates (e g deer), bear. fish, lagamorphs rodents etc Environmental samples include en ironmental material such as surface matter soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention. The invention contemplates detecting bacterial toxin by a competitive immunoassay method that utilizes recombinant toxin VTl and toxin VT2 proteins, antibodies raised against recombinant bacterial toxin proteins. A fixed amount of the recombinant toxin proteins are immobilized to a solid suppoπ (e.g., a microtiter plate) followed by the addition of a biological sample suspected of containing a bacterial toxin. The biological sample is first mixed with affinity-purified or PEG fractionated antibodies directed against the recombinant toxin protein. A reporter reagent is then added which is capable of detecting the presence of antibody bound to the immobilized toxin protein. The reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used to identify the presence of the reporter substance. If toxin is present in the sample, this toxin will compete with the immobilized recombinant toxin protein for binding to the anti- recombinant antibody thereby reducing the signal obtained following the addition of the repoπer reagent. A control is employed where the antibody is not mixed w ith the sample. This gives the highest (or reference) signal. The invention also contemplates detecting bacterial toxin by a "sandwich" immunoassay method that utilizes antibodies directed against recombinant bacterial toxin proteins. Affinity-purified antibodies directed against recombinant bacterial toxin proteins are immobilized to a solid support (e.g.. microtiter plates). Biological samples suspected of containing bacterial toxins are then added followed by a washing step to remove substantially all unbound antitoxin. The biological sample is next exposed to the reporter substance, w hich binαs to antitoxin and is then washed free of substantially all unbound reporter substance. The repoπer substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used to identify the presence of the reporter substance. Identification of the repoπer substance in the biological tissue indicates the presence of the bacterial toxin. It is also contemplated that bacterial toxin be detected by pouring liquids (e.g.. soups and other fluid foods and feeds including nutritional supplements for humans and other animals) over immobilized antibody which is directed against the bacterial toxin. It is contemplated that the immobilized antibody will be present in or on such suppoπs as caπridges. columns, beads, or any other solid suppoπ medium. In one embodiment, following the exposure of the liquid to the immobilized antibody, unbound toxin is substantially removed by washing. The liquid is then exposed to a reporter substance which detects the presence of bound toxin. In a preferred embodiment the reporter substance is an enzyme, fluorescent dye, or radioactive compound attached to an antibody which is directed against the toxin (i.e., in a "sandwich" immunoassay). It is also contemplated that the detection system will be developed as necessary (e.g.. the addition of enzyme substrate in enzyme systems: observation using fluorescent light for fluorescent dye systems: and quantitation of radioactivity for radioactive systems). EXPERIMENTAL The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. In the disclosure which follows, the following abbreviations apply: °C (degrees Centigrade): rpm (revolutions per minute); BSA (bovine serum albumin): ELISA (enzyme- linked immunosorbent assay): IgG (immunoglobulin G): IgY (immunoglobulin Y): IP (intraperitoneal): SC (subcutaneous): H 2 O (water): HCI (hydrochloric acid): LD I ()() (lethal dose for 100%o of experimental animals): aa (amino acid): HPLC (high performance liquid chromatography): Kda (kilodaltons): gm (grams): μg (micrograms): mg (milligrams); ng (nanograms): μl (microliters): ml (milliliters): mm (millimeters); nm (nanometers): μm (micrometer): M (molar): mM (millimolar): MW (molecular weight): sec (seconds): min(s) (minute/minutes): hr(s) (hour/hours): MgCL (magnesium chloride): NaCl (sodium chloride): Na-CO, (sodium carbonate): OD :80 (optical density at 280 nm); OD 6l , (l (optical density at 600 nm): PAGE (polyacrylamide gel electrophoresis): PBS [phosphate buffered saline ( 150 mM NaCl. 10 mM sodium phosphate buffer. pH 7.2)]: PEG (polyethylene glycol): SDS (sodium dodecyl sulfate): Tris (tris(hydroxymethyl)aminomethane): vv/v (weight to volume): v/v (volume to volume): Amicon (Amicon. Inc.. Beverly, MA): Amresco (Amresco. Inc.. Solon. OH); ATCC (American Type Culture Collection. Rockville. MD): BBL (Baltimore Biologies Laboratory, (a division of Becton Dickinson). Cockeysville. MD); Becton Dickinson (Becton Dickinson Labware. Lincoln Park. NJ); BioRad (BioRad. Richmond. CA): Biotech (C-C Biotech Corp.. Povvay. CA): Charles River (Charles River Laboratories. Wilmington. MA): Falcon (e.g. Baxter Healthcare Corp.. McGaw Park. IL and Becton Dickinson): Fisher Biotech (Fisher Biotech. Springfield. NJ); GIBCO (Grand Island Biologic Company/BRL. Grand Island. NY): Mallinckrodt (a division of Baxter Healthcare Corp.. McGaw Park. IL): Millipore (Millipore Corp., Marlborough, MA); New England Biolabs (New England Biolabs. Inc.. Beverly. MA); Novagen (Novagen, Inc.. Madison. WI); Pharmacia (Pharmacia. Inc.. Piscataway, NJ); Qiagen (Qiagen. Chatsworth, CA): Showdex (Showa Denko America. Inc.. New York. NY); Sigma (Sigma Chemical Co., St. Louis. MO): RIBI (RIBI Immunochemical Research Inc.. Hamilton. MT); Accurate Chemical and Scientific Corp. (Accurate Chemical and Scientific Corp.. Hicksville, NY); Kodak (Eastman-Kodak. Rochester. NY); and Stratagene (Stratagene. La Jolla. CA). When a recombinant protein is described in the specification it is referred to in a short-hand manner by the amino acids in the toxin sequence present in the recombinant protein rounded to the nearest 10. The specification gives detailed construction details for all recombinant proteins such that one skilled in the art will know precisel which amino acids are present in a given recombinant protein. The first set of Examples (Examples 1 -5) was designed to develop an antidote to £ coli O157:H7 verotoxins and evaluate its effectiveness in vitro and in vivo. In the first experiments, high titer verotoxin antibodies were generated in laying hens hyperimmunized with chemically detoxified and/or native verotoxins. These Laying hens were immunized with either recombinant £. coli O157:H7 VTl or VT2 (rVTl and rVT2) treated with glutaraldehyde and mixed with adjuvant. Next, toxin-reactive polyclonal antibodies were isolated by bulk fractionation from egg yolks pooled from hyperimmunized hens. Large quantities of polyclonal antibodies (IgY) were harvested from resulting eggs using a two-step polyethylene glycol fractionation procedure. Third, the immunoreactivity and yields of VT IgY were analyzed by analytical immunochemical methods (e.g., enzyme immunoassay (EIA) and Western blotting). El A and Western blot analysis showed that the resulting egg preparations contained high titer IgY that reacted with both the immunizing and the heterologous toxins (i.e., rVTl IgY reacted against both rVTl and rVT2. and vice versa). Fouπh. VT neutralization potency was analyzed in vitro using a Vero cytotoxicity assay. Vero cytotoxicity of rVTl and rVT2 could be completely inhibited by VT IgY. These antibodies also demonstrated substantial verotoxin cross-neutralization. Fifth, the efficacy of passively administered avian verotoxin antibodies in preventing the lethal effects of verotoxin poisoning was assessed in a mouse disease model. Toxin neutralizing antibodies were administered by parenteral dosing regimens to assess the most effective strategy for therapeutic intervention. Efficacy of v erotoxin antibodies was demonstrated using multiple muπne disease models. In these experiments, antibodies prevented both the morbidity and lethality of homologous and heterologous toxins using a toxin/antitoxin premix format: mice infected orally with a lethal dose of v lable £ coli O157:H7 were protected from both morbidity and lethality w hen treated parenterally four hours post-infection with either rVTl or rVT2 antibodies: and mice giv en a lethal dose of £ coli O91 :H21 (a paπicularly virulent strain which only produces VT2c. a VT2 structural variant) and treated parenterally up to 10 hours later with rVTl IgY administered parenterally were protected from both morbidity and lethality EXAMPLE 1 TOXIN ANALYSIS AND IMMUNIZATION Purified recombinant £ coli O157:H7 verotoxins. rVTl and rVT2. were obtained from Denka Sieken Co.. Ltd. (Tokyo. Japan). Toxin genes were isolated, inserted into expression plasmids. and expressed in £ coli Recombinant proteins were then purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE Sephacry 1 and hydroxyapatite. and gel filtration chromatography by the supplier. Upon receipt, toxins were analyzed to verify identity, purity and toxicity, as described below A. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Samples of each toxin (2 μg) were heat-denatured in a buffer containing SDS and β- mercaptoethanol followed by electrophoresis on 10-20% gradient gels ( Bio-Rad. Richmond. CA). Resolved polypeptide bands were visualized using the silv er stain procedure of C R Merπl. ei al.. "Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins." Science 21 1 1437- 1438 ( 1981 ) VTl and VT2 are each composed of subunit A and multiple copies of subunit B Subunit A is often nicked into fragments A l and A2 which are linked by a disulfide bridge As shown in Figure I . when separated by SDS-PAGE in the presence of β-mercaptoethanol. rVT l resolv ed into 3 bands that corresponded to subunit A (-31 Kda). fragment A l (-27 Kda) and a mixture of subunit B and fragment A2 (-4 Kda) Similarly . rVT2 resolved into subunit A (-33 Kda). fragment A l (-27 Kda) and a mixture of subunit B and fragment A2 (-8 Kda) (Figure 1 ) In this Figure. rVTl is in Lane 1. and rVT2 is in Lane 2. the positions ot molecular weight markers (Kda) are shown at the left. VT component poly peptides are identified at the right. These results are consistent with previous reports of VTl and VT2 purified from naturally occurring toxigenic strains (V. V. Padhye et ai. "Purification and Physicochemical Properties of a Unique Vero Cell Cytotoxin From Escherichia coli O157:H7." Biochem. Biophys. Res. Commun.. 139: 424-430 [1986]; and F. B. Kittel et ai. "Characterization and inactivation of verotoxin 1 produced by Escherichia coli O157:H7." J. Agr. Food Chem.. 39: 141-145 [1991]). B. High Performance Liquid Chromatography (HPLC). Chromatography was performed at room temperature (RT) under isocratic conditions using a Waters 510 HPLC pump. Eluted protein was measured using a W aters 490E programmable multi-wavelength detector (Millipore Corp.. Milford. MA). The VT's were separated on an 8 x 300 mm (ID) Shodex KW803 column, using 10 mM sodium phosphate. 0.15 M NaCl. pH 7.4 (phosphate buffered saline [PBS]) as the mobile phase at a flow rate of 1 ml/min. The purity of non-denatured rVT ' s was assessed by HPLC. As shown in the chromatographs in Figure 2, each toxin eluted at approximately 10 min. as a single absorbance peak at 280 nm. By integration of the area under each peak, the rVT ' s were shown to be >99% pure. C. Vero Cell Cytotoxicity Assay. Cytotoxic activity of rVTl and rVT2 was assessed using modified procedures of Padhye. et al. (V. V. Padhye et ai. "Purification and Physicochemical Properties of a Unique Vero Cell Cytotoxin From Escherichia coli O157:H7." Biochem. Biophys. Res. Commun.. 139: 424-430 [1986]). and McGee. et al, (Z. A. McGee. et ai. "Local induction of tumor necrosis factor as molecular mechanism of mucosal damage by gonococci." Microbial Pathogenesis 12: 333-341 [1992]). Microtiter plates (96 well. Falcon. Microtest III) were inoculated with approximately 1 x 10 4 Vero cells (ATCC. CCL81 ) per well ( 100 μl) and incubated overnight at 37°C in the presence of 5% CO : to form Vero cell monolayers. rVTl and rVT2 solutions were serially diluted in Medium 199 supplemented with 5% fetal bovine serum (Life Technologies. Grand Island. NY), added to each well of the microtiter plates and incubated at 37°C for 18-24 hrs. Adherent (viable) cells were stained with 0.2% crystal violet (Mallinckrodt) in 2% ethanol. Excess stain was rinsed away and the stained cells were solubilized by adding 100 μl of 1% SDS to each well. Absorbance of each well was measured at 570 nm. and the percent cytotoxicity of each test sample was calculated using the following formula: % Vero Cytotoxicity = [ 1 - (Absorbance Sample/Absorbance Control)] x 100 To determine whether the rVT ' s possessed potency equivalent to published cytotoxicity values, a Vero cell cytotoxicity assay was performed (Figure 3). Between 0.01-10.000 pg of either rVTl or rVT2 was added to Vero cells. The amounts of rVT causing 50% cell death (CD ?() ), as calculated by second degree polynomial curve fitting were 0.97 pg and 1.5 pg. for rVTl and rVT2. respectively. These results are consistent with CD<„ values reported previously for naturally occurring VTl and VT2 in the range 1 -35 pg and 1-25 pg. respectively (M. Petric et ai. Purification and biological properties of Escherichia coli verocytotoxin." FEMS Microbiol. Lett.. 41 : 63-68 [ 1987]: V. L. Tesh. et ai . "Comparison of relative toxicities of Shiga-Like toxins Type I and Type II for mice." Infect. Immun.. 61 : 3392-3402 [1993]: N. Dickie et ai . "Purification of an Escherichia coli Serogroup O 157:H7 verotoxin and its detection in Noπh American hemorrhagic colitis isolates," J. Clin. Microbiol.. 27: 1973-1978 [1989]; and U. Kongmuang. et ai. "A simple method for purification of Shiga or Shiga-Like toxin from Shigella dysenteriae and Escherichia coli O 157:H7 by immunoaffinity chromatography." FEMS Microbiol. Lett.. 48: 379-383 [ 1987]). It has been observed that toxicity is lost with storage, explaining why higher amounts of toxin were used in the neutralization assays described below. D. Mouse Lethal Dose Determination. To verify rVTl and rVT2 toxicity. male (20-22 g) CD- I mice were injected intraperitoneally with varying amounts of rVTl or rVT2 in 200 μL phosphate buffer. Doses were selected based on published LD 5n values for VTl and VT2 in CD- I mice. To minimize the sacrifice of live animals, a full statistical toxin LD 50 was not determined. Mice were observed for morbidit and moπality over 7-day period. Fuπher confirmation of rVT toxicity was obtained from mouse lethality experiments (Table 2). Mice were injected intraperitoneally with varying amounts of either rVTl or rVT2 and observed 7 days for mortality. Within 72-120 hrs. post-injection, all of the mice died from 100 ng of rVTl or 10 ng of rVT2. respectively * . This lethality study served as a verification of expected toxicity but not as a statistical determination of LD 50 . Nonetheless, these results were consistent with toxicity studies which reported LD 50 values in CD-I mice of 0.4-2.0 μg for purified VTl and 0.001-1.0 μg for purified VT2 (V. L. Tesh. et ai . "Comparison of relative toxicities of Shiga-Like toxins Type I and Type II for mice." Infect. Immun.. 61 : 3392-3402 [1993]; and A. D. O'Brien, and G. D. LaVeck. "Purification and characterization of Shigella dysenteriae 1 -like toxin produced by Escherichia coli." Infect. Immun. 40: 675-683 [1983]). Table 2. Lethality of rVTl in CD-I Mice ng VTl Injected Survivors/Total Hours Post-Injection 7/7 24 ± 2 100 5/7 48 ± 2 0/7 72 ± 2 7/7 24 ± 2 10 7/7 48 ± 2 7/7 72 ± 2 6/6 24 ± 2 1.0 6/6 48 ± 2 6/6 72 ± 2 Table 3. Lethality of rVT2 in CD-I Mice ng VT2 Injected Survivors/Total Hours Post-Injection 3/6 48 ± 2 10 2/6 72 ± 2 0/6 120 ± 2 5/6 48 ± 2 1.0 4/6 72 ± 2 0/6 120 ± 2 6/6 48 ± 2 0.1 6/6 72 ± 2 6/6 120 ± 2 The recombinant toxins used in these studies thus appeared to contain protein components and toxicities consistent with literature reports for native toxins. Based on these structural and functional analyses, the rVT's were considered suitable as antigens to generate specific avian antibodies. E. Antigen Preparation. Lyophilized samples. rVTl and rVT2 were received and each was reconstituted with 2.5 mL of deionized water to a final concentration of 100 μg/ml in phosphate buffer. To form a toxoid. the solutions were then treated with 0.4% glutaraldehyde (Mallinckrodt) at 4°C overnight and stored at -20°C thereafter. When needed, toxoid was thawed and mixed 5: 1 (volume:volume) with GERBU adjuvant (C. C. Biotech Corporation. Poway. CA). White Leghorn laying hens were injected subcutaneously with 25 μg of either rVTl or rVT2 toxoid in adjuvant at 2-3 week intervals. EXAMPLE 2 PEG EXTRACTION OF EGG YOLK ANTIBODY Hyperimmune eggs were collected after 3 immunizations with toxoid. Egg yolks were separated from whites, pooled according to their immunogen group and blended with 4 volumes of 10 mM sodium phosphate. 150 mM NaCl. pH 7.4 (PBS). Polyethylene glycol 8000 (PEG) (Amresco. Solon. OH) was then added to a final concentration of 3.5% and the mixture centrifuged at 10,000 x g for 10 min. to remove the precipitated lipid fraction. IgY- rich supernatant was filtered through cheesecloth and PEG was again added to a final concentration of 12%. The solution was centrifuged as above and the resulting supernatant discarded. The IgY pellet was then dissolved in PBS to either the original ( I X PEG IgY) or V* of the original (4X PEG IgY) yolk volume, filtered through a 0.45 μ membrane and stored at 4°C. EXAMPLE 3 ANTITOXIN IMMUNOASSAYS A. Enzyme Immunoassay (EIA). EIA was used to monitor antibody responses during the immunization course. Wells of 96-well Pro-Bind microtiter plates (Falcon, through Scientific Products. McGaw Park. IL) were each coated with 1 μg of rVT's (not toxoid) in PBS overnight at 2-8°C. Wells were washed 3 times with PBS containing 0.05% Tween-20 (PBS-T) to remove unbound antigen, and the remaining protein binding sites were blocked with PBS containing 1 mg/ml BSA for 60 min. at room temperature (RT). IgY. diluted in PBS, was then added to the wells and incubated for 1 hr. at 37°C. Wells were washed as before to remove unbound primary antibody and incubated for 1 hr. at 37°C with alkaline phosphatase-conjugated rabbit-anti- chicken IgG (Sigma Chemical Company. St. Louis. MO) diluted 1 : 1000 in PBS-T. Wells were again washed and 1 mg/ml /. -nitrophenyl phosphate (Sigma Chemical Company. St. Louis. MO) in 50 mM Na 2 CO ; 10 mM MgC pH 9.5 was added and allowed to incubate at RT Phosphatase activity was detected by absorbance at 410 nm using a Dynatech MR700 microtiter plate reader. Laying Leghorn hens were immunized as described above (Example 1. pan E). using glutaraldehyde-treated rVT ' s. Following several immunizations, eggs were collected and IgY harvested by PEG fractionation. Figures 4 and 5 show rVTl or rVT2 specific antibody responses detected using EIA at dilutions of the original yolk IgY concentration of 1 :30.000 and 1 :6.000. respectively. IgY fractionated similarly from unimmunized hens (i. e.. preimmune antibody) did not react with either antigen at test dilutions above 1 :50. Although these EIA results indicate significant antibody responses, prior experience with other toxin antigens has shown that optimization of immunization regimens, including increasing the amount of antigen, can yield titers in excess of 1 : 100.000 (B. S. Thalley. el ai . "Development of an Avian Antitoxin to Type A Botulinum Neurotoxin." in Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. B. R. DasGupta. (ed.) [Plenum Press. New York. 1993] pp. 467-472). As may be expected due to their structural homology and consistent with previous repoHs (e.g., \'. V. Padhye et ai. "Production and characterization of monoclonal antibodies to verotoxins 1 and 2 from Escherichia coli O157:H7." J. Agr. Food Chem.. 39: 141-145 [1989]; S. C. Head et ai . "Purification and characterization of verocytotoxin 2." FEMS Microbiol. Lett.. 51 : 21 1-216 [1988]; and N. C. Strockbine et ai. "Characterization of Monoclonal Antibodies against Shiga-Like Toxin from Escherichia coli." Infect. Immun.. 50: 695-700 [1985]). Figures 4 and 5 also demonstrate that antibodies generated against one toxin cross-reacted in vitro with the other toxin. B. Western Blot Analysis. Western blots (Figure 6) performed to determine the reactivity of rVT antibodies against constituent VT polypeptides showed that rVTl and rVT2 antibodies reacted with subunit A and fragment A l of either toxin, and with subunit B and fragment A2 of rVT l only. In this Figure. Panel A contains preimmune IgY. Panel B contains rVT l IgY. and Panel C contains rVT2 IgY. Lane 1 in each panel contains rVTl (2μg) and Lane 2 contains rVT2 (2 μg). Preimmune IgY was largely nonreactive to either rVT. Both rVT IgY preparations, however, failed to react with subunit B and fragment A2 of rVT2. Some explanations for this lack of measurable reactivity might include poor immunogenicity. denaturation of the immunogen during glutaraldehyde treatment, loss of conformational epitopes due to detergent or reducing agent, or poor transfer to nitrocellulose. To resolve the high and low molecular weight components. 2 μg each of rVTl and rVT2 were separated by SDS-PAGE (described above) and then transferred to nitrocellulose paper using the Milliblot-SDE system (Millipore. Medford. MA) according to the manufacturer ' s instructions. Paper strips were stained temporarily with Ponceau S (Sigma Chemical Company. St. Louis. MO) to visualize the polypeptides and then blocked overnight in PBS containing 5% dry milk. Each strip was agitated gently in IgY diluted in PBS-T for 2 hrs. at RT. Strips were each washed with three changes of PBS-T to remove unbound primary- antibody and incubated for 2 hrs. at RT with goat anti-chicken alkaline phosphatase (Kirkegaard and Perry. Gaithersburg. MD) diluted 1 :500 in PBS-T containing 1 mg/ml BSA. The blots were washed as before and rinsed in 50 mM Na : CO„ pH 9.5. Strips were submerged in alkaline-phosphatase substrate (5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (Kirkegaard and Perry) until sufficient signal was observed. Color development was stopped by flooding the blots with water. EXAMPLE 4 IN VITRO TOXIN NEUTRALIZATION: VERO CELL ASSAY IgY neutralization of rVTl and rVT2 was assessed using the modified Vero cytotoxicity assay described above (Example 1. pan C). Various concentrations of IgY. diluted in Medium 199 supplemented with 5% fetal bovine serum (GIBCO). were mixed with sufficient toxin to cause 50% cell death and allowed to incubate at 37°C for 60 minutes. These toxin antibody mixtures were then added to Vero cell-coated microtiter plate wells according to the procedure described above (Example 1. part C). The toxin neutralization capacity of the rVT antibodies was analyzed first using a Vero cell toxicity assay. The results in Figure 7 show that rVTl IgY neutralized completely the cytotoxic activity of rVTl at an endpoint dilution of 1/320. Furthermore. rVT2 IgY neutralized the heterologous rVTl toxin, but at a higher endpoint concentration. In a similar experiment (see Figure 8). rVTl and rVT2 antibodies were each able to neutralize rVT2 at equivalent endpoint dilutions. This strong cross-neutralization correlates with the observed strong cross-reactivity of VTl IgY with VT2 A seen on Western blots (Figure 6). These results show that IgY antibodies are able to neutralize effectively VT cytotoxicity and that the antibodies can cross-neutralize structurally-related heterologous toxins. EXAMPLE 5 TOXIN NEUTRALIZATION: MOUSE ASSAYS A. Toxin Challenge Model. IgY in PBS was premixed with a lethal dose of toxin (as determined above) and injected intraperitoneally into male CD-I (20-22 gm) mice. Mice were observed for a 7-day period for signs of intoxication such as ruffled fur. huddling and disinclination to mov e. followed by hind leg paralysis, rapid breathing and death. Untreated, infected mice usually died within 12 hrs. after signs of severe illness (i.e . within 48-72 hrs. post-injection). Once it was demonstrated that rVT antibodies were able to neutralize rVT cytotoxicity in vitro, protection experiments were next performed in mice. First, animals were challenged with rVT premixed with rVT IgY to determine whether toxin lethality could be neutralized under conditions optimal for antigen/antibody reaction. Tables 4 and 5 show that antibodies premixed with the homologous toxin (e.g., rVTl with rVTl IgY) prevented lethality of rVT. Preimmune IgY was unable to neutralize either toxin in these studies. Table 4 Neutralization of rVTl Using rVT IgY 100 ng rVT2 Premixed* Survivors/Total P Preimmune Antibody 0/12 rVTl Antibody 12/12 < 0.001 rVT2 Antibody 12/12 < 0.001 Toxin was pre-mixed with IgY and incubated for 1 hour at room temperature prior to administration. Table 5 Neutralization of rVT2 Using rVT IgY 10 ng rVTl Premixed* Survivors/Total P Preimmune Antibody 0/12 rVTl Antibody 12/12 < 0.001 rVT2 Antibody 12/12 < 0.001 *Toxin was pre-mixed with IgY and incubated for 1 hour at room temperature prior to administration. Antibodies premixed with the heterologous toxin (e.g.. rVT2 with rVTl IgY) also prevented lethality in vivo. These data are in contrast to previous observations where rabbit polyclonal antibodies generated against either toxin were cross-reactive with the heterologous toxin by EIA and Western blot, but were unable to neutralize the heterologous toxin in either Vero cell cytotoxicity and mouse lethality assays (S. C. Head, et ai . "Serological differences between verocytotoxin 2 and Shiga-like toxin II." Lancet ii: 751 [1988]: S. C. Head et ai . "Purification and characterization of verocvtotoxin 2." FEMS Microbiol. Lett.. 51 : 21 1 -216 [1988]; N. C. Strockbine et ai. "Characterization of Monoclonal Antibodies against Shiga- Like Toxin from Escherichia coli." Infect. Immun., 50: 695-700 [ 1985]: and V. V. Padhye et ai . "Purification and Physicochemical Properties of a Unique Vero Cell Cy totoxin From Escherichia coli O157:H7." Biochem. Biophys. Res. Commun.. 139: 424-430 [1986]). However. Head et ai. showed that VT2 B-subunit specific monoclonal antibodies neutralized VTl weakly in a Vero cytotoxicity assay (S. C. Head, et al . "Serological differences between verocytotoxin 2 and Shiga-like toxin II." Lancet ii: 751 [1988]). In a report by Donohue-Rolfe. et ai . a VT2 B subunit-specific monoclonal antibody neutralized both VTl and VT2 completely in a Hela cytotoxicity assay (A. Donohue-Rolfe et ai . "Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized PI glycoprotein and production of cross reactive monoclonal antibodies." Infect. Immun.. 57: 3888-3893 [1989]). These results showed for the first time complete cross-neutralization in Vero cell cytotoxicity and mouse lethality assays, revealing that VTl and VT2 do indeed share common neutralizing epitopes. These results may indicate that hens generate different antibody specificities as compared to mammals, and/or that differences in immunization methods might have maintained the immunogenicity of conformational epitopes necessary for cross- neutralization. Nonetheless, this cross-neutralization suggests that IgY antibodies may contain the range of reactivities essential for an effective antitoxin. B. Viable organism infection model. Streptomycin-resistant £ coli O 157:H7 (strain 933 cu-rev) or £ coli O9 UH21 ( strain B2I 1 ) (both kindly provided by Dr. Alison O'Brien. Dept. of Microbiology and Immunology. Uniformed Services University of the Health Sciences. Bethesda. MD) were used in a murine infection model described by Wadolkowski. et al. (E. A. Wadolkowski et ai. "Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7." Infect. Immun.. 58: 2438-2445 [1990]). Organisms were grown in Luria broth and incubated overnight at 37°C in an Environ Shaker (Lab Line. Melrose Park. IL) (T. Maniatis et ai . Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor. N. Y., [1982]). Bacterial suspensions were centrifuged at 6700 x g for 5 minutes. The resulting pellet was then washed twice with sterile PBS and resuspended in sterile 20% (w/v) sucrose. Five to 8 week-old male CD-I mice were provided drinking water containing 5 mg/ml streptomycin sulfate ad libitum for 24 hrs. Food and water were then withheld for another 16-18 hrs. after which mice were challenged orally with 10"' streptomycin-resistant £. coli O157:H7 or O91.Η21. Mice were housed individually and permitted food and water containing 5 mg/ml streptomycin sulfate. IgY was injected intraperitoneally at varying times post- infection and animals observed for both morbidity and moπality for 10 days. To monitor bacterial colonization in animals, 1 gram of feces was collected. homogenized, and plated onto MacConkey agar medium (Difco Laboratories. Detroit. MI) containing 100 μg/ml streptomycin and incubated at 37°C as described by Wadolkowski. et al. (E. A. Wadolkowski et ai, "Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7." Infect. Immun.. 58: 2438-2445 [1990]). The serotype of £. coli O157:H7. 933 cu-rev excreted in feces was confirmed by slide agglutination with O- and H-specific antisera (Difco Laboratories. Detroit. MI). Kidneys were removed from experimental animals and fixed in 10% buffered neutral formalin. Sections of parafilm-embedded tissue were stained with hematoxy lin and eosin (General Medical Laboratories. Madison. WI) and examined by light microscopy. All tissue sections were coded to avoid bias before microscopic examination to determine renal pathology. The toxin neutralization ability of rVT IgY was further studied using a streptomycin- treated CD-I mouse infection model. This model was chosen because it produces definitive systemic pathology and reproducible mortality. In contrast to previous studies by Wadolkowski. et al. (E. A. Wadolkowski et ai. "Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin Type II." Infect. Immun.. 58: 3959-3965 [ 1 90]). where mice were given subunit-specific monoclonal antibodies prior to infection, the mice in this study were inoculated orally with 2 x 10 10 viable £ coli O157:H7 (strain 933 cu-rev) and treated with rVT IgY 4 hrs. follow ing inoculation. Fecal cultures showed that 10 -10 8 challenge organisms per gram of feces were shed throughout the course of the experiment, thus confirming that infection was established. Tables 6 and 7 show that animals treated with either rVTl or rVT2 IgY were protected from lethality caused by infection (p<0.01 and p<0.001. respectively) and that preimmune IgY failed to provide protection to the mice. Table 6 Protection of Mice From E. coli O157:H7 With rVTl IgY IgY Treatment Survivors/Total P Morbidity/Total Preimmune Antibody 0/5 5/5 rVTl Antibody 9/10 < 0.01 1/10 *IgY was administered intraperitoneally 4 hours following infection, and once daily for davs thereafter. Table 7 Protection of Mice From E. coli O157:H7 With rVT2 IgY IgY Treatment Survivors/Total P Morbidity/Total Preimmune Antibody 0/6 6/6 rVT2 Antibody 10/10 < 0.005 0/10 *IgY was administered intraperitoneally 4 hours following infection, and once daily for 10 days thereafter. Renal histopathology (i * ee Figure 9) of the control (preimmune IgY) animals showed dilation, degeneration and renal tubular necrosis with no glomerular damage. This is consistent with previous reports showing that renal tubular involvement occurs predominantly in this streptomy cin-treated mouse infectivity model (E. A. Wadolkowski el al . "Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin Type II." Infect. Immun.. 58: 3959-3965 [1990]). Importantly , none of the survivors exhibited similar signs of morbidity though treated with IgY 4 hrs. after infection (.see Figure 9). Furthermore, avian antibodies generated against rVTl were able to prevent both oπality and morbidity in a mouse model where VT2 alone is implicated in the pathogenesis and lethality of £ coli O157:H7 strain 933 cu-rev (E. A. Wadolkowski et al . "Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin Type II." Infect. Immun.. 58: 3959-3965 [ 1990]). To assess the broader utility of the IgY verotoxin antibodies in treating VTEC- associated disease, the mouse infectivity study was performed using a more v irulent VTEC serotype known to produce VT2c — a structural variant of VT — but not VTl (S. W. Lindgren et ai, "Virulence of enterohemorrhagic Escherichia coli O91 :H21 clinical isolates in an orally infected mouse model." Infect. Immun.. 61 : 3832-3842 [ 1993]). Mice were inoculated orally with 5 x 10 9 E. coli O91 :H21 (strain B2F1 ) and treated subsequently with IgY. Notably, the heterologous rVTl IgY protected strongly against the lethal effects of the VT2c structural variant, even when administered as long as 10 hrs. following infection (Table 8). Ten hours was the longest treatment window tested in this study. Only 1 of the 8 animals treated with rVTl IgY died (p <0.02). and those that survived showed no oven signs of renal histopathology (i.e.. acute bilateral tubular necrosis). It can thus be concluded that rVTl IgY completely neutralized toxicity of VT2c. indicating its potential as a therapeutic for at least one other pathogenic VTEC. Table 8 Protection of Mice From E. coli O91 :H21 With rVTl IgY IgY Treatment Survivors/Total P Morbidity/Total Preimmune Antibody 0/7 7/7 rVTl Antibody 7/8 < 0.02 1/8 *IgY was administered intraperitoneally 10 hours following infection, and once daily for 8 days thereafter. These Examples highlight several impoπant findings supporting the feasibility of using verotoxin antitoxin. First, polyclonal IgY generated against either VTl or VT2 from £. coli O157:H7. cross-reacted with and fully cross-neutralized the toxicity of the non-immunizing toxin both in vitro and in vivo. Second, recombinant toxins fully neutralized the toxicity of naturally-occurring toxins produced by £ coli O157:H7 during the course of infection. Third, antibodies generated against rVTl from £ coli O157:H7 could prevent morbidity and moπality in mice infected orally with lethal doses of £. coli O91 :H21. a particularly virulent strain which only produces VT2c. suggesting their utility in preventing systemic sequelae. Because VTl is identical to Shiga-toxin (A. D. O ' Brien et ai, "Shiga and Shiga-like toxins. Microbial Rev.. 51 : 206-220 [1987]), VT antibodies may also be useful in preventing complications stemming from Shigella dysenteriae infection. Finally, animals treated w ith VT IgY were protected against both death and kidney damage when treated as long as 10 hrs after infection, supposing the hypothesis that a window for antitoxin interv ention exists These studies strongly suppoπ the use of parenterally-administered. toxin-specific IgY as a antitoxin to prevent life-threatening complications associated with £ t oll 0157 H7 and other VTEC infections. It is contemplated that this approach would be most useful in preventing HUS and other complications when administered after the onset of bloody diarrhea and before the presentation of sy stemic disease The VT IgY developed in these studies were shown to react with and neutralize both recombinant and naturally-occurring VT The antibody titers as measured bv EIA are indicative of reasonable antibody production in the hen, however much higher production levels can be obtained with larger immunizing doses The results from these Examples clearly demonstrate the teasibihtv and prov ide the experimental basis for development of an avian antidote for £ coli 0157 H7 v erotoxins suitable for use in humans In contrast to previous reports showing that rabbit polyclonal VTl and VT2 antibodies cross-reacted, but did not cross-neutralize the heterologous toxin in Vero cytotoxicity or in mouse lethality studies (e g V V Padhye et ul Production and characterization of monoclonal antibodies to verotoxins 1 and 2 from Escherichia coli O157 H7." J Agr Food Chem . 39 141 -145 [1989], S C Head et al . "Purification and characterization of verocytotoxin 2." FEMS Microbiol Lett . 51 21 1-216 [ 1988]. and N C Strockbine et al "Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli." Infect Immun . 50 695-700 [1985]). these data prov ide the first demonstration of cross-neutralization in vivo Antibodies against one toxin neutralized completely the heterologous toxin in both Vero cytotoxicity and mouse lethality assay s Both rVl 1 and rVT2 antibodies also prevented morbidity (as assessed by renal histopathology ) and mortality in mice infected with lethal doses of £ coli 0157 H7 - the etiologic agent in 90% of the documented cases of hemolv tic uremic syndrome (HUS) in the S (P M Griffin and R V Tauxe. "The epidemiology of infections caused by Escherichia colt 0157 H7. other enterohemorrhagic £ coli. and the associated hemolvtic uremic syndrome Epidemiol Rev 13 60 [ 1990]) With at least two other VTEC serotypes known to cause HUS. the finding that rVTl antibodies neutralized a VT2 variant produced by £ coli 091 H21 suggests that av ian polyclonal antibodies may provide an effective antidote against other v erotoxin- producing £ coli These data also show for the first time, that antibodies may be administered after infection and still protect against morbidity and morta tv EXAMPLE 6 EXPRESSION OF TOXIN GENES The previous Examples clearly showed that avian polyclonal antibodies to recombinant toxins protected animals infected with verotoxigenic £. coli. This Example includes expression of toxin genes (A and B subunits alone and together as whole toxins) in suitable prokaryotic expression systems to achieve high levels of VT antigen production. The sequence of the toxin gene has been determined (see e.g.. M.P. Jackson et ai. "Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933." 44: 109 [1987]). The coding regions of the A and B subunits of VT-1 are listed in SEQ ID NOS: l and 3. respectively. The corresponding amino acid sequence of the A and B subunits of the VT-1 toxin are listed in SEQ ID NOS:2 and 4, respectively. The coding regions of the A and B subunits of VT-2 are listed in SEQ ID NOS:5 and 7. respectively. The corresponding amino acid sequence of the A and B subunits of the VT-2 toxin are listed in SEQ ID NOS:6 and 8. respectively. In addition. SEQ ID NOS:9 and 10 list the sequences which direct the expression of a poly-cistronic RNA capable of directing the translation of both the A and B subunits from the VT-1 and VT-2 genes, respectively. In choosing a strategy for recombinant VT antigen production, there are three primary technical factors to consider. First, the appropriate VT antigen components representing the spectrum of toxin epitopes encountered in nature must be utilized. Second, the protein antigens must be expressed at sufficient levels and purity to enable immunization and large- scale antibody purification. Third, the neutralizing epitopes must be preserv ed in the immunogen and immunoabsorbant. Approaches that offer the greatest promise for high level expression of periplasmically localized, native, affinity-tagged proteins were developed. Figure 10 shows the fusion constructs of VT components and affinity tags. A. Expression of affinity-tagged C-terminal constructs. The VTl and VT2 A and B subunits (SEQ ID NOS: l. 3. 5 and 7) are cloned into the pET-23b vector (Novagen). This vector is designed to allow expression of native proteins containing C-terminal poly-His tags. The vector utilizes a strong T7 polymerase promoter to drive high level expression of target proteins. The methionine initiation codon is engineered to contain a unique Nde\ restriction enzyme site (CATATG). The VTl and VT2 genes are engineered to conveπ the signal sequence methionine codon into a h'del site utilizing PCR mutagenesis. PCR primers were designed which contain the sequence GCCAT fused to the first 20-24 bases of the genes (starting at the ATG start codon of the signal tag; SEQ ID NOS: 12-19. see Table below). Upon PCR amplification, the 5 ' start codon of each gene is converted to an Ndel site, compatible with the pET-23 vector-encoded A'del site, allowing cloning of the amplified genes into the vector without the addition of vector-encoded amino acids. Primers containing the C-terminal 7 codons of each gene (21 bases) fused to the sequence CTCGAGCC were synthesized, in order to add a C-terminal poly-His tag to each gene. The underlined bases are an Xho\ site, that is compatible with the Λ77oI site of the pET-23 vector. These primers precisely delete the native stop codons. and when cloned into the pET-23 vector, add a C-terminal extension of "LeuGluHisHisHisHisHisHis" (SEQ ID NO: 1 1). The following table lists the primer pairs are utilized to create PCR fragments containing the A and B subunits derived from VT-1 and VT-2 toxin genes suitable for insertion into the pET-23b vector. Table 9 Primers Toxin Gene and Subunit N-terminal Primer C-terminal Primer VT-1 Subunit A SEQ ID NO: 12 SEQ ID NOT3 VT-1 Subunit B SEQ ID NO: 14 SEQ ID NO: 15 VT-2 Subunit A SEQ ID NO: 16 SEQ ID NO: 17 VT-2 Subunit B SEQ ID NO: 18 SEQ ID NO: 19 VT-1 Subunits A and B SEQ ID NO: 12 SEQ ID NO: 15 VT-2 Subunits A and B SEQ ID NO: 16 SEQ ID NO: 19 Thus, utilizing PCR amplification with the above modified N- and C-terminal primers, the A and B subunits of VTl and VT2 are expressed as proteins containing an 8 amino acid C-terminal extension bearing an poly-histidine affinity tag. The amino acid sequence of the histidine-tagged VT-1 A subunit produced by expression from the pET-23b vector is listed in SEQ ID NO:21 (the associated DNA sequence is listed in SEQ ID NO:20): the amino acid sequence of the histidine-tagged VT-1 B subunit is listed in SEQ ID NO:23 (the associated DNA sequence is listed in SEQ ID NO:22); the amino acid sequence of the histidine-tagged VT-2 A subunit is listed in SEQ ID NO:25 (the associated DNA sequence is listed in SEQ ID NO:24); the amino acid sequence of the histidine-tagged VT-2 B subunit is listed in SEQ ID NO:27 (the associated DNA sequence is listed in SEQ ID NO:26). Both subunits may be expressed from a single expression constructs by utilizing SEQ ID NOS: 12 and 15 to prime synthesis of the VT-1 toxin gene and SEQ ID NOS: 16 and 19 to prime synthesis of the VT-2 toxin gene. The resulting PCR products are cleaved with Ndel and Xlio . as described for the cloning of the subunit genes into the pET-23b vector. Expression of the A and B subunits from such an expression v ector. results in the expression of a native A subunit and a his-tagged B subunit. As the A and B subunits assemble into a complex, the presence of the his-tag on only the B subunit is sufficient to allow purification of the holotoxin on metal chelate columns as described below. The proofreading Pfu polymerase (Stratagene) is utilized for PCR amplification to reduce the error rate during amplification. Genomic DNA from an £. coli O157:H7 strain is utilized as template DNA. Following the PCR, the amplification products are digested with Ndel and Xho\ and cloned into the pCR-Script SK cloning v ehicle (Stratagene) to permit DNA sequence analysis of the amplified products. The DNA sequence analysis is performed to ensure that no base changes are introduced during amplification. Once the desired clones are identified by DNA sequencing, the inserts are then excised utilizing Ndel and Λ7.øl. and cloned into a similarly cut pET-23b vector to create the expression constructs. According to the published sequences, neither the VTl nor VT2 genes contain either of these restriction sites. The poly-His-tagged proteins produced by expression of the VT- 1 and VT-2 gene sequences in the pET-23b constructs are then purified by IMAC. This method uses metal- chelate affinity chromatography to purify native or denatured proteins which have histidine tails (see e.g., K. J. Petty, "Metal-Chelate Affinity Chromatography." in Current Protocols in Molecular Biology, Supplement 24. Unit 10.1 IB [1993]). B. Expression of Toxin Containing N-terminal Affinity Tags Two expression systems, pMal-p2 and pFLAG-1 are utilized to attach an N-terminal affinity tag to the A subunits from the VT-1 and VT-2 toxins. MBP-tagged constructs. To construct A chains containing the maltose binding protein (MBP) at the N-terminus of the A subunit. PCR amplified gene products are cloned into the pMal-p2 vector (New England Biolabs) as C-terminal fusions to a periplasmically-secreted version of the MBP. The MBP selectively binds to amylose resins and serves as an affinity tag on the MBP/A subunit fusion protein. The pMal-p2 vector contains an engineered factor Xa cleavage site, which permits the removal of the affinity tag (i.e.. MBP) from the fusion protein after purification. The MBP/A subunit fusions are generated as follows. The VTl and VT2 A subunits are PCR-amplified utilizing the following DNA primers. SEQ ID NOS:28-31 : SEQ ID NOS:28 and 29 comprise the 5 ' and 3' primers, respectively, for the amplification of the VTl A subunit; SEQ ID NOS:30 and 31 comprise the 5' and 3' primers, respectively, for the amplification of the VT2 A subunit. In both cases, the 5' or N-terminal primer contains the sequence CGGAATTC fused to the first codon of the mature polypeptide (rather than the start of the signal peptide. since the MBP signal peptide is utilized). These 5 " primers contain an engineered £coRl site that is not contained internally in either gene, that is compatible with the EcoRl site of the pMal-p2 vector. The 3' or C-terminal primers incorporate an Xhol site as described above for the generation of the His-tagged toxins, but in this case, the 3 " primer is designed to include the natural termination codon of the A subunits. The genes are amplified, cloned into pCR-Script SK, and sequenced as described above. The inserts are then excised with £coRI and Xhol, and cloned into Ec-oRI/Stf/I-cleaved pMal-p2 vector (Sail and Xhol sites are compatible). This construct allows expression and secretion of the VTl and VT2 A subunit genes as C-terminal fusions with MBP. The amino acid sequence of the MBP/VT-1A fusion protein is listed in SEQ ID NO:33 (the associated DNA sequence is listed in SEQ ID NO:32). The amino acid sequence of the MBP/VT-2.A fusion protein is listed in SEQ ID NO:35 (the associated DNA sequence is listed in SEQ ID NO.34). The resulting fusion proteins are then affinity purified on an amylose column and the bound fusion protein is eluted under mild conditions by competition with maltose. The MBP N-terminal-tagged A subunits are cleaved with factor Xa and the MBP is removed by chromatography on an amylose column. The resulting A subunits which contain a 4 amino acid N-terminal extension are then used as immunogens. Flag tag constructs. In an alternative embodiment, the VTl and VT2 A subunit genes are engineered to contain the "flag tag" through the use of the pFLAG-1 vector system. The flag tag is located between the O pA secretion signal sequence and the authentic N- terminus of the target protein in the pFlag-1 vector. To construct N-terminal flag-tagged A chains, the EcoRl/Xhol A subunit PCR fragments (generated as described above for the MBP fusion proteins) are cloned into identically cleaved pFlag-1 vector (Eastman-Kodak), to produce an expression construct utilizing the OmpA signal peptide for secretion of A subunit fusion proteins containing the flag peptide at the N-terminus. After secretion, the periplasmic protein contains the N-terminal 8 amino acid flag tag, followed by 4 vector-encoded amino acids fused to the recombinant A subunit. The amino acid sequence of the flag tag/VT- 1 A subunit fusion protein is listed in SEQ ID NO:37 (the associated DNA sequence is listed in SEQ ID NO:36). The amino acid sequence of the flag tag/VT-2 A subunit fusion protein is listed in SEQ ID NO:39 (the associated DNA sequence is listed in SEQ ID NO:38). The flag tag fusion proteins are then purified by immunoaffinity chromatography utilizing a calcium-dependent monoclonal antibody (Antiflag Ml : Eastman-Kodak). Mild elution of purified protein is achieved by chelating the calcium in the column buffer with ethylenediamine tetraacetic acid (EDTA). C. Evaluation of fusion construct expression. The fusion constructs described above are expressed in £. coli strain BL21. or T7 polymerase-containing derivatives [e.g.. BL21(DE3). BL21(DE3) pLysS. BL21(DE3)pLysE] (Novagen) for pET plasmids. and periplasmically-secreted recombinant protein purified by affinity chromatography. Recombinant proteins are analyzed for correct conformation by testing the following parameters: a) It is believed that the B subunit must associate into pentamers to be conformationally correct. This is assessed by reducing and native SDS-PAGE analyses of native and chemically-cross-linked proteins and sizing HPLC: b) It is believed that a properly folded A subunit is expected to retain its native enzymatic activity. This is tested by its capacity to inhibit protein synthesis in an in vitro toxicity assay; c) It is believed that in vitro toxicity of assembled recombinant holotoxin is compared to commercially available holotoxins to determine whether recombinant A and B subunits can assemble into functional holotoxin. The purified N-terminal-tagged A subunits (after cleavage and purification from MBP or untreated flag-tagged proteins) are combined in vitro with the corresponding B chains, and their toxicity evaluated utilizing a quantitative microtiter cytotoxicity assay, such as that described by M.K. Gentry and M. Dalrymple, "Quantitative Microtiter Cytotoxicity Assay for Shigella Toxin." J. Clin. Microbiol., 12:361-366 (1980). SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: OPHIDIAN PHARMACEUTICALS, INC. (ii) TITLE OF INVENTION: TREATMENT FOR VEROTOXIN-PRODUCING E. COLI (iii) NUMBER OF SEQUENCES: 39 (iv) CORRESPONDENCE ADDRESS : (A) ADDRESSEE: MEDLEN & CARROLL (B) STREET: 220 MONTGOMERY STREET, SUITE 2200 (C) CITY: SAN FRANCISCO (D) STATE: CALIFORNIA (E) COUNTRY: UNITED STATES OF AMERICA (F) ZIP: 94104 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version si.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNE /AGENT INFORMATION: (A) NAME: CARROLL, PETER G. (B) REGISTRATION NUMBER: 32,837 (C) REFERENCE/DOCKET NUMBER: OPHD-02171 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 705-8410 (B) TELEFAX: (415) 397-8336 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 945 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..945 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : ATG AAA ATA ATT ATT TTT AGA GTG CTA ACT TTT TTC TTT GTT ATC TTT 48 Met Lys He He He Phe Arg Val Leu Thr Phe Phe Phe Val He Phe 1 5 10 15 TCA GTT AAT GTG GTG GCG AAG GAA TTT ACC TTA GAC TTC TCG ACT GCA 96 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 25 30 AAG ACG TAT GTA GAT TCG CTG AAT GTC ATT CGC TCT GCA ATA GGT ACT 144 Lys Thr Tyr Val Asp Ser Leu Asn Val He Arg Ser Ala He Gly Thr 35 ' 40 45 CCA TTA CAG ACT ATT TCA TCA GGA GGT ACG TCT TTA CTG ATG ATT GAT 192 Pro Leu Gin Thr He Ser Ser Gly Gly Thr Ser Leu Leu Met He Asp 50 55 60 AGT GGC TCA GGG GAT AAT TTG TTT GCA GTT GAT GTC AGA GGG ATA GAT 240 Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly He Asp 65 70 75 80 GCA GAG GAA GGG CGG TTT AAT AAT CTA CGG CTT ATT GTT GAA CGA AAT 288 Ala Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu He Val Glu Arg Asn 85 90 95 AAT TTA TAT GTG ACA GGA TTT GTT AAC AGG ACA AAT AAT GTT TTT TAT 336 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tvr 100 105 110 CGC TTT GCT GAT TTT TCA CAT GTT ACC TTT CCA GGT ACA ACA GCG GTT 384 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 ACA TTG TCT GGT GAC AGT AGC TAT ACC ACG TTA CAG CGT GTT GCA GGG 432 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Glv 130 135 140 ATC AGT CGT ACG GGG ATG CAG ATA AAT CGC CAT TCG TTG ACT ACT TCT 480 He Ser Arg Thr Gly Met Gin He Asn Arg His Ser Leu Thr Thr Ser 145 150 155 160 TAT CTG GAT TTA ATG TCG CAT AGT GGA ACC TCA CTG ACG CAG TCT GTG 526 Tyr Leu AΞD Leu Met Ser His Ser Gly Thr Ser Leu Thr Gin Ser Val 165 170 175 GCA AGA GCG ATG TTA CGG TTT GTT ACT GTG ACA GCT GAA GCT TTA CGT 576 Ala Arg Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 TTT CGG CAA ATA CAG AGG GGA TTT CGT ACA ACA CTG GAT GAT CTC AGT 624 Phe Arg Gin He Gin Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 GGG CGT TCT TAT GTA ATG ACT GCT GAA GAT GTT GAT CTT ACA TTG AAC 672 Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 TGG GGA AGG TTG AGT AGC GTC CTG CCT GAC TAT CAT GGA CAA GAC TCT 720 Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His Gly Gin Asp Ser 225 " 230 235 240 GTT CGT GTA GGA AGA ATT TCT TTT GGA AGC ATT AAT GCA ATT CTG GGA 768 Val Arg Val Glv Arg He Ser Phe Gly Ser He Asn Ala He Leu Gly 245 250 255 AGC GTG GCA TTA ATA CTG AAT TGT CAT CAT CAT GCA TCG CGA GTT GCC 816 Sei Val Ala Leu He Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 AGA ATG GCA TCT GAT GAG TTT CCT TCT ATG TGT CCG GCA GAT GGA AGA 864 Arg Met Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 ' 285 GTC CGT GGG ATT ACG CAC AAT AAA ATA TTG TGG GAT TCA TCC ACT CTG 912 Val Arg Gly He Thr His Asn Lys He Leu Trp Asp Ser Ser Thr Leu 290 ' 295 300 GGG GCA ATT CTG ATG CGC AGA ACT ATT AGC AGT 945 Gly Ala He Leu Met Arg Arg Thr He Ser Ser 305 310 315 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 315 ammo acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : Met Lys He He He Phe Arg Val Leu Thr Phe Phe Phe Val He Phe 1 5 10 15 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 25 30 Lys Thr Tyr Val Asp Ser Leu Asn Val He Arg Ser Ala He Gly Thr 35 40 45 Pro Leu Gin Thr He Ser Ser Gly Gly Thr Ser Leu Leu Met He Asp 50 55 60 Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly He Asp 65 70 75 80 Ala Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu He Val Glu Arg Asn 85 90 95 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100 105 110 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Gly 130 - 135 140 He Ser Arg Thr Gly Met Gin He Asn Arg His Ser Leu Thr Thr Ser 145 150 155 160 Tyr Leu Asp Leu Met Ser His Ser Gly Thr Ser Leu Thr Gin Ser Val 165 170 175 Ala Arg Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 Phe Arg Gin He Gin Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His Gly Gin Asp Ser 225 230 235 240 Val Arg Val Gly Arg He Ser Phe Gly Ser He Asn Ala He Leu Gly 245 250 255 Ser Val Ala Leu He Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 Arg Met Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285 Val Arg Gly He Thr His Asn Lys He Leu Trp Asp Ser Ser Thr Leu 290 295 300 Gly Ala He Leu Met Arg Arg Thr He Ser Ser 305 310 315 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 267 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..267 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : ATG AAA AAA ACA TTA TTA ATA GCT GCA TCG CTT TCA TTT TTT TCA GCA 48 Met Lys Lys Thr Leu Leu He Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 15 AGT GCG CTG GCG ACG CCT GAT TGT GTA ACT GGA AAG GTG GAG TAT ACA 96 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 20 25 30 AAA TAT AAT GAT GAC GAT ACC TTT ACA GTT AAA GTG GGT GAT AAA GAA 144 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 35 40 45 TTA TTT ACC AAC AGA TGG AAT CTT CAG TCT CTT CTT CTC AGT GCG CAA 192 Leu Phe Thr Asn Arg Trp Asn Leu Gin Ser Leu Leu Leu Ser Ala Gin 50 55 60 ATT ACG GGG ATG ACT GTA ACC ATT AAA ACT AAT GCC TGT CAT AAT GGA 240 He Thr Gly Met Thr Val Thr He Lys Thr Asn Ala Cys His Asn Gly 65 70 75 80 GGG GGA TTC AGC GAA GTT ATT TTT CGT * 267 Gly Gly Phe Ser Glu Val He Phe Arg 85 (2) INFORMATION FOR SEQ ID NO:4 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 89 ammo acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Lys Lys Thr Leu Leu He Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 15 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 20 25 30 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 35 40 45 Leu Phe Thr Asn Arg Trp Asn Leu Gin Ser Leu Leu Leu Ser Ala Gin 50 55 60 He Thr Gly Met Thr Val Thr He Lys Thr Asn Ala Cys His Asn Gly 65 70 75 80 Gly Gly Phe Ser Glu Val He Phe Arg 85 (2) INFORMATION FOR SEQ ID NO: 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 954 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..954 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATG AAG TGT ATA TTA TTT AAA TGG GTA CTG TGC CTG TTA CTG GGT TTT 48 Met Lys Cys He Leu Phe Lys Trp Val Leu Cys Leu Leu Leu Gly Phe 1 5 10 15 TCT TCG GTA TCC TAT TCC CGG GAG TTT ACG ATA GAC TTT TCG ACC CAA 96 Ser Ser Val Ser Tyr Ser Arg Glu Phe Thr He Asp Phe Ser Thr Gin 20 25 30 CAA AGT TAT GTC TCT TCG TTA AAT AGT ATA CGG ACA GAG ATA TCG ACC 144 Gin Ser Tyr Val Ser Ser Leu Asn Ser He Arg Thr Glu He Ser Thr 35 40 45 CCT CTT GAA CAT ATA TCT CAG GGG ACC ACA TCG GTG TCT GTT ATT AAC 192 Pro Leu Glu His He Ser Gin Gly Thr Thr Ser Val Ser Val He Asn 50 55 60 CAC ACC CAC GGC AGT TAT TTT GCT GTG GAT ATA CGA GGG CTT GAT GTC 240 His Thr His Gly Ser Tyr Phe Ala Val Asp He Arg Gly Leu Asp Val 65 70 75 80 TAT CAG GCG CGT TTT GAC CAT CTT CGT CTG ATT ATT GAG CAA AAT AAT 288 Tyr Gin Ala Arg Phe Asp His Leu Arg Leu He He Glu Gin Asn Asn 85 90 95 TTA TAT GTG GCA GGG TTC GTT AAT ACG GCA ACA AAT ACT TTC TAC CGT 336 Leu Tyr Val Ala Gly Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg 100 105 110 TTT TCA GAT TTT ACA CAT ATA TCA GTG CCC GGT GTG ACA ACG GTT TCC 384 Phe Ser Asp Phe Thr His He Ser Val Pro Gly Val Thr Thr Val Ser 115 120 125 ATG ACA ACG GAC AGC AGT TAT ACC ACT CTG CAA CGT GTC GCA GCG CTG 432 Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Ala Leu 130 135 140 GAA CGT TCC GGA ATG CAA ATC AGT CGT CAC TCA CTG GTT TCA TCA TAT 480 Glu Arg Ser Gly Met Gin He Ser Arg His Ser Leu Val Ser Ser Tyr 145 150 155 160 CTG GCG TTA ATG GAG TTC AGT GGT AAT ACA ATG ACC AGA GAT GCA TCC 528 Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser 165 170 175 AGA GCA GTT CTG CGT TTT GTC ACT GTC ACA GCA GAA GCC TTA CGC TTC 576 Arg Ala Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe 180 185 190 AGG CAG ATA CAG AGA GAA TTT CGT CAG GCA CTG TCT GAA ACT GCT CCT 624 Arg Gin He Gin Arg Glu Phe Arg Gin Ala Leu Ser Glu Thr Ala Pro 195 200 205 GTG TAT ACG ATG ACG CCG GGA GAC GTG GAC CTC ACT CTG AAC TGG GGG 672 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly 210 215 220 CGA ATC AGC AAT GTG CTT CCG GAG TAT CGG GGA GAG GAT GGT GTC AGA 720 Arg He Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg 225 230 235 240 GTG GGG AGA ATA TCC TTT AAT AAT ATA TCA GCG ATA CTG GGG ACT GTG 768 Val Gly Arg He Ser Phe Asn Asn He Ser Ala He Leu Gly Thr Val 245 250 255 GCC GTT ATA CTG AAT TGC CAT CAT CAG GGG GCG CGT TCT GTT CGC GCC 816 Ala Val He Leu Asn Cys His His Gin Gly Ala Arg Ser Val Arσ Ala 260 265 270 GTG AAT GAA GAG AGT CAA CCA GAA TGT CAG ATA ACT GGC GAC AGG CCT 864 Val Asn Glu Glu Ser Gin Pro Glu Cys Gin He Thr Gly Asp Arσ Pre 275 280 285 GTT ATA AAA ATA AAC AAT ACA TTA TGG GAA AGT AAT ACA GCT GCA GCG 912 Val He Lys He Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala 290 295 300 TTT CTG AAC AGA AAG TCA CAG TTT TTA TAT ACA ACG GGT AAA 954 Phe Leu Asn Arg Lys Ser Gin Phe Leu Tyr Thr Thr Gly Lys 305 310 315 (2) INFORMATION FOR SEQ ID NO:6 : ti) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 ammo acids (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Lys Cys He Leu Phe Lys Trp Val Leu Cys Leu Leu Leu Gly Phe 1 * 5 10 15 Ser Ser Val Ser Tyr Ser Arg Glu Phe Thr He Asp Phe Ser Thr Gin 20 25 30 Gin Ser Tyr Val Ser Ser Leu Asn Ser He Arg Thr Glu He Ser Thr 35 40 45 Pro Leu Glu His He Ser Gin Gly Thr Thr Ser Val Ser Val He Asn 50 55 60 His Thr His Gly Ser Tyr Phe Ala Val Asp He Arg Gly Leu Asp Val 65 70 75 80 Tyr Gin Ala Arg Phe Asp His Leu Arg Leu He He Glu Gin Asn Asn 85 90 95 Leu Tyr Val Ala Gly Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg 100 105 110 Phe Ser Asp Phe Thr His He Ser Val Pro Gly Val Thr Thr Val Ser 115 120 125 Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Ala Leu 130 " 135 140 Glu Arg Ser Gly Met Gin He Ser Arg His Ser Leu Val Ser Ser Tyr 145 ' 150 155 160 Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser 165 170 175 Arg Ala Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe 180 185 190 Arg Gin He Gin Arg Glu Phe Arg Gin Ala Leu Ser Glu Thr Ala Pro 195 200 205 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly 210 215 220 Arg He Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg 225 230 235 240 Val Gly Arg He Ser Phe Asn Asn He Ser Ala He Leu Gly Thr Val 245 250 255 Ala Val He Leu Asn Cys His His Gin Gly Ala Arg Ser Val Arg Ala 260 265 270 Val Asn Glu Glu Ser Gin Pro Glu Cys Gin He Thr Gly Asp Arg Pro 275 280 285 Val He Lys He Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala 290 295 300 Phe Leu Asn Arg Lys Ser Gin Phe Leu Tyr Thr Thr Gly Lys 305 310 315 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 267 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..267 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 : ATG AAG AAG ATG TTT ATG GCG GTT TTA TTT GCA TTA GCT TCT GTT AAT 48 Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Val Asn 1 5 10 15 GCA ATG GCG GCG GAT TGT GCT AAA GGT AAA ATT GAG TTT TCC AAG TAT 96 Ala Met Ala Ala Asp Cys Ala Lys Gly Lys He Glu Phe Ser Lys Tyr 20 25 30 AAT GAG GAT GAC ACA TTT ACA GTG AAG GTT GAC GGG AAA GAA TAC TGG 144 Asn Glu Asp Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp 35 40 45 ACC AGT CGC TGG AAT CTG CAA CCG TTA CTG CAA AGT GCT CAG TTG ACA 192 Thr Ser Arg Trp Asn Leu Gin Pro Leu Leu Gin Ser Ala Gin Leu Thr 50 55 60 GGA ATG ACT GTC ACA ATC AAA TCC AGT ACC TGT GAA TCA GGC TCC GGA 240 Glv Met Thr Val Thr He Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly 6 * 5 70 75 8 * 0 TTT GCT GAA GTG CAG TTT AAT AAT GAC 267 Phe Ala Glu Val Gin Phe Asn Asn Asp 85 (2) INFORMATION FOR SEQ ID NO:8 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Val Asn 1 5 10 15 Ala Met Ala Ala Asp Cys Ala Lys Gly Lys He Glu Phe Ser Lys Tyr 20 25 30 Asn Glu Asp Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp 35 40 45 Thr Ser Arg Trp Asn Leu Gin Pro Leu Leu Gin Ser Ala Gin Leu Thr 50 55 60 Gly Met Thr Val Thr He Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly 65 70 75 80 Phe Ala Glu Val Gin Phe Asn Asn Asp 85 (2) INFORMATION FOR SEQ ID NO: 9 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1241 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : ATGAAAATAA TTATTTTTAG AGTGCTAACT TTTTTCTTTG TTATCTTTTC AGTTAATGTG 60 GTGGCGAAGG AATTTACCTT AGACTTCTCG ACTGCAAAGA CGTATGTAGA TTCGCTGAAT 120 GTCATTCGCT CTGCAATAGG TACTCCATTA CAGACTATTT CATCAGGAGG TACGTCTTTA 180 CTGATGATTG ATAGTGGCTC AGGGGATAAT TTGTTTGCAG TTGATGTCAG AGGGATAGAT 240 GCAGAGGAAG GGCGGTTTAA TAATCTACGG CTTATTGTTG AACGAAATAA TTTATATGTG 300 ACAGGATTTG TTAACAGGAC AAATAATGTT TTTTATCGCT TTGCTGATTT TTCACATGTT 360 ACCTTTCCAG GTACAACAGC GGTTACATTG TCTGGTGACA GTAGCTATAC CACGTTACAG 420 CGTGTTGCAG GGATCAGTCG TACGGGGATG CAGATAAATC GCCATTCGTT GACTACTTCT 480 TATCTGGATT TAATGTCGCA TAGTGGAACC TCACTGACGC AGTCTGTGGC AAGAGCGATG 540 TTACGGTTTG TTACTGTGAC AGCTGAAGCT TTACGTTTTC GGCAAATACA GAGGGGATTT 600 CGTACAACAC TGGATGATCT CAGTGGGCGT TCTTATGTAA TGACTGCTGA AGATGTTGAT 660 CTTACATTGA ACTGGGGAAG GTTGAGTAGC GTCCTGCCTG ACTATCATGG ACAAGACTCT 720 GTTCGTGTAG GAAGAATTTC TTTTGGAAGC ATTAATGCAA TTCTGGGAAG CGTGGCATTA 780 ATACTGAATT GTCATCATCA TGCATCGCGA GTTGCCAGAA TGGCATCTGA TGAGTTTCCT 840 TCTATGTGTC CGGCAGATGG AAGAGTCCGT GGGATTACGC ACAATAAAAT ATTGTGGGAT 900 TCATCCACTC TGGGGGCAAT TCTGATGCGC AGAACTATTA GCAGTTGAAC AGGGGGTAAA 960 TAAAGGAGTT AAGCATGAAA AAAACATTAT TAATAGCTGC ATCGCTTTCA TTTTTTTCAG 1020 CAAGTGCGCT GGCGACGCCT GATTGTGTAA CTGGAAAGGT GGAGTATACA AAATATAATG 1080 ATGACGATAC CTTTACAGTT AAAGTGGGTG ATAAAGAATT ATTTACCAAC AGATGGAATC 1140 TTCAGTCTCT TCTTCTCAGT GCGCAAATTA CGGGGATGAC TGTAACCATT AAAACTAATG 1200 CCTGTCATAA TGGAGGGGGA TTCAGCGAAG TTATTTTTCG T 1241 ( 2 ) INFORMATION FOR SEQ ID NO : 10 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 1235 base pairs ( B ) TYPE : nucleic acid ( C ) STRANDEDNESS : double ( D ) TOPOLOGY : l inear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATGAAGTGTA TATTATTTAA ATGGGTACTG TGCCTGTTAC TGGGTTTTTC TTCGGTATCC 60 TATTCCCGGG AGTTTACGAT AGACTTTTCG ACCCAACAAA GTTATGTCTC TTCGTTAAAT 120 AGTATACGGA CAGAGATATC GACCCCTCTT GAACATATAT CTCAGGGGAC CACATCGGTG 180 TCTGTTATTA ACCACACCCA CGGCAGTTAT TTTGCTGTGG ATATACGAGG GCTTGATGTC 240 TATCAGGCGC GTTTTGACCA TCTTCGTCTG ATTATTGAGC AAAATAATTT ATATGTGGCA 300 GGGTTCGTTA ATACGGCAAC AAATACTTTC TACCGTTTTT CAGATTTTAC ACATATATCA 360 GTGCCCGGTG TGACAACGGT TTCCATGACA ACGGACAGCA GTTATACCAC TCTGCAACGT 420 GTCGCAGCGC TGGAACGTTC CGGAATGCAA ATCAGTCGTC ACTCACTGGT TTCATCATAT 480 CTGGCGTTAA TGGAGTTCAG TGGTAATACA ATGACCAGAG ATGCATCCAG AGCAGTTCTG 540 CGTTTTGTCA CTGTCACAGC AGAAGCCTTA CGCTTCAGGC AGATACAGAG AGAATTTCGT 600 CAGGCACTGT CTGAAACTGC TCCTGTGTAT ACGATGACGC CGGGAGACGT GGACCTCACT 660 CTGAACTGGG GGCGAATCAG CAATGTGCTT CCGGAGTATC GGGGAGAGGA TGGTGTCAGA 720 GTGGGGAGAA TATCCTTTAA TAATATATCA GCGATACTGG GGACTGTGGC CGTTATACTG 780 AATTGCCATC ATCAGGGGGC GCGTTCTGTT CGCGCCGTGA ATGAAGAGAG TCAACCAGAA 840 TGTCAGATAA CTGGCGACAG GCCTGTTATA AAAATAAACA ATACATTATG GGAAAGTAAT 900 ACAGCTGCAG CGTTTCTGAA CAGAAAGTCA CAGTTTTTAT ATACAACGGG TAAATAAAGG 960 AGTTAAGCAT GAAGAAGATG TTTATGGCGG TTTTATTTGC ATTAGCTTCT GTTAATGCAA 1020 TGGCGGCGGA TTGTGCTAAA GGTAAAATTG AGTTTTCCAA GTATAATGAG GATGACACAT 1080 TTACAGTGAA GGTTGACGGG AAAGAATACT GGACCAGTCG CTGGAATCTG CAACCGTTAC 1140 TGCAAAGTGC TCAGTTGACA GGAATGACTG TCACAATCAA ATCCAGTACC TGTGAATCAG 1200 GCTCCGGATT TGCTGAAGTG CAGTTTAATA ATGAC 1235 (2) INFORMATION FOR SEQ ID NO: 11: !ι) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 am o acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (11) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11. Leu Glu His His His His His His 1 5 (2) INFORMATION FOR SEQ ID NO:12- (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12. GCCATATGAA AATAATTATT TTTAGAGTG 29 (2) INFORMATION FOR SEQ ID NO:13. (l) SEQUENCE CHARACTERISTICS. (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid JO STRANDEDNESS. single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13. GGCTCGAGAC TGCTAATAGT TCTGCGCAT 29 (2) INFORMATION FOR SEQ ID NO:14: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid C) STRANDEDNESS: single D< TOPOLOGY linear Ui) MOLECULE TYPE: DNA (genomic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCCATATGAA AAAAACATTA TTAATAGC 28 (2) INFORMATION FOR SEQ ID NO: 15: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs \3) TYPE- nucleic acid (C) STRANDEDNESS: single iD) TOPOLOGY: linear Ui) MOLECULE TYPE. DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 GGCTCGAGAC GAAAAATAAC TTCGCTGAA 29 (2) INFORMATION FOR SEQ ID NO:16: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (11) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCCATATGAA GTGTATATTA TTTAAATGG 29 (2) INFORMATION FOR SEQ ID NO:17: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17. GGCTCGAGTT TACCCGTTGT ATATAAAAAC 30 .2) INFORMATION FOR SEQ ID NO:18: U) SEQUENCE CHARACTERISTICS. (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY, linear Ui) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CGCATATGAA GAAGATGTTT ATGGCG 26 (2) INFORMATION FOR SEQ ID NO:19: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS. single (D) TOPOLOGY linear Ui) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGCTCGAGGT CATTATTAAA CTGCACTTC 29 (2) INFORMATION FOR SEQ ID NO:20. U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 969 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) Ux) FEATURE: (A) NAME/KEY- CDS (B) LOCATION: 1 .969 ixi) SEQUENCE DESCRIPTION- SEQ ID NO:20 ATG AAA ATA ATT ATT TTT AGA GTG CTA ACT TTT TTC TTT GTT ATC TTT 48 Met Lys He He He Phe Arg Val Leu Thr Phe Phe Phe Val He Phe 1 5 10 15 TCA GTT AAT GTG GTG GCG AAG GAA TTT ACC TTA GAC TTC TCG ACT GCA 96 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 25 30 AAG ACG TAT GTA GAT TCG CTG AAT GTC ATT CGC TCT GCA ATA GGT ACT 144 Lys Thr Tyr Val Asp Ser Leu Asn Val He Arg Ser Ala He Gly Thr 35 40 45 CCA TTA CAG ACT ATT TCA TCA GGA GGT ACG TCT TTA CTG ATG ATT GAT 192 Pro Leu Gin Thr He Ser Ser Gly Gly Thr Ser Leu Leu Met He Asp 50 55 60 AGT GGC TCA GGG GAT AAT TTG TTT GCA GTT GAT GTC AGA GGG ATA GAT 240 Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly He Asp 65 70 75 80 GCA GAG GAA GGG CGG TTT AAT AAT CTA CGG CTT ATT GTT GAA CGA AAT 288 Ala Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu He Val Glu Arg Asn 85 90 95 AAT TTA TAT GTG ACA GGA TTT GTT AAC AGG ACA AAT AAT GTT TTT TAT 336 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100 105 110 CGC TTT GCT GAT TTT TCA CAT GTT ACC TTT CCA GGT ACA ACA GCG GTT 384 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 ACA TTG TCT GGT GAC AGT AGC TAT ACC ACG TTA CAG CGT GTT GCA GGG 432 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Gly 130 135 140 ATC AGT CGT ACG GGG ATG CAG ATA AAT CGC CAT TCG TTG ACT ACT TCT 480 He Ser Arg Thr Gly Met Gin He Asn Arg His Ser Leu Thr Thr Ser 145 150 155 160 TAT CTG GAT TTA ATG TCG CAT AGT GGA ACC TCA CTG ACG CAG TCT GTG 528 Tyr Leu Asp Leu Met Ser His Ser Gly Thr Ser Leu Thr Gin Ser Val 165 170 175 GCA AGA GCG ATG TTA CGG TTT GTT ACT GTG ACA GCT GAA GCT TTA CGT 576 Ala Arg Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 TTT CGG CAA ATA CAG AGG GGA TTT CGT ACA ACA CTG GAT GAT CTC AGT 624 Phe Arg Gin He Gin Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 GGG CGT TCT TAT GTA ATG ACT GCT GAA GAT GTT GAT CTT ACA TTG AAC 672 Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 TGG GGA AGG TTG AGT AGC GTC CTG CCT GAC TAT CAT GGA CAA GAC TCT 720 Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His Gly Gin Asp Ser 225 230 235 240 GTT CGT GTA GGA AGA ATT TCT TTT GGA AGC ATT AAT GCA ATT CTG GGA 768 Val Arg Val Gly Arg He Ser Phe Gly Ser He Asn Ala He Leu Gly 245 250 255 AGC GTG GCA TTA ATA CTG AAT TGT CAT CAT CAT GCA TCG CGA GTT GCC 816 Ser Val Ala Leu He Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 AGA ATG GCA TCT GAT GAG TTT CCT TCT ATG TGT CCG GCA GAT GGA AGA 864 Arg Met Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285 GTC CGT GGG ATT ACG CAC AAT AAA ATA TTG TGG GAT TCA TCC ACT CTG 912 Val Arg Gly He Thr His Asn Lys He Leu Trp Asp Ser Ser Thr Leu 290 295 300 GGG GCA ATT CTG ATG CGC AGA ACT ATT AGC AGT CTC GAG CAC CAC CAC 960 Gly Ala He Leu Met Arg Arg Thr He Ser Ser Leu Glu His His His 305 310 315 320 CAC CAC CAC 969 His His His (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 323 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Lys He He He Phe Arg Val Leu Thr Phe Phe Phe Val He Phe 1 5 10 15 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 25 30 Lys Thr Tyr Val Asp Ser Leu Asn Val He Arg Ser Ala He Gly Thr 35 40 45 Pro Leu Gin Thr He Ser Ser Gly Gly Thr Ser Leu Leu Met He Asp 50 55 60 Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly He Asp 65 70 75 80 Ala Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu He Val Glu Arg Asn 85 90 95 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100 " 105 110 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Gly 130 135 140 He Ser Arg Thr Glv Met Gin He Asn Arg His Ser Leu Thr Thr Ser 145 " 150 155 160 Tyr Leu Asp Leu Met Ser His Ser Gly Thr Ser Leu Thr Gin Ser Val 165 170 175 Ala Arg Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 Phe Arg Gin He Gin Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 Trp Gly Arσ Leu Ser Ser Val Leu Pro Asp Tyr His Gly Gin Asp Ser 225 " 230 235 240 Val Arg Val Gly Arg He Ser Phe Gly Ser He Asn Ala He Leu Gly 245 250 255 Ser Val Ala Leu He Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 Arg Met Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285 Val Arg Gly He Thr His Asn Lys He Leu Trp Asp Ser Ser Thr Leu 290 295 300 Gly Ala He Leu Met Arg Arg Thr He Ser Ser Leu Glu His His His 305 310 315 320 (2) INFORMATION FOR SEQ ID NO:22: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 294 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) Ux) FEATURE: (A) NAME/KEY. CDS (B) LOCATION. 1..294 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22. ATG AAA AAA ACA TTA TTA ATA GCT GCA TCG CTT TCA TTT TTT TCA GCA 48 Met Lys Lys Thr Leu Leu He Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 15 AGT GCG CTG GCG ACG CCT GAT TGT GTA ACT GGA AAG GTG GAG TAT ACA 96 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 20 25 30 AAA TAT AAT GAT GAC GAT ACC TTT ACA GTT AAA GTG GGT GAT AAA GAA 144 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 35 40 45 TTA TTT ACC AAC AGA TGG AAT CTT CAG TCT CTT CTT CTC AGT GCG CAA 192 Leu Phe Thr Asn Arg Trp Asn Leu Gin Ser Leu Leu Leu Ser Ala Gin 50 55 60 ATT ACG GGG ATG ACT GTA ACC ATT AAA ACT AAT GCC TGT CAT AAT GGA 240 He Thr Gly Met Thr Val Thr He Lys Thr Asn Ala Cys His Asn Gly 65 * 70 75 80 GGG GGA TTC AGC GAA GTT ATT TTT CGT CTC GAG CAC CAC CAC CAC CAC 288 Gly Gly Phe Ser Glu Val He Phe Arg Leu Glu His His His His His 85 90 95 CAC TG 294 His (2) INFORMATION FOR SEQ ID NO:23: U) SEQUENCE CHARACTERISTICS: (A) LENGTH 97 amino acids (B) TYPE: amino acid (D) TOPOLOGY linear Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION SEQ ID NO:23 Met Lys Lys Thr Leu Leu He Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 15 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 20 25 30 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 35 40 45 Leu Phe Thr Asn Arg Trp Asn Leu Gin Ser Leu Leu Leu Ser Ala Gin 50 55 60 He Thr Gly Met Thr Val Thr He Lys Thr Asn Ala Cys His Asn Gly 65 70 75 80 Gly Gly Phe Ser Glu Val He Phe Arg Leu Glu His His His His His 85 90 95 His (2) INFORMATION FOR SEQ ID NO:24: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 981 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..981 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: ATG AAG TGT ATA TTA TTT AAA TGG GTA CTG TGC CTG TTA CTG GGT TTT 48 Met Lys Cys He Leu Phe Lys Trp Val Leu Cys Leu Leu Leu Gly Phe 1 5 10 15 TCT TCG GTA TCC TAT TCC CGG GAG TTT ACG ATA GAC TTT TCG ACC CAA 96 Ser Ser Val Ser Tyr Ser Arg Glu Phe Thr He Asp Phe Ser Thr Gin 20 25 30 CAA AGT TAT GTC TCT TCG TTA AAT AGT ATA CGG ACA GAG ATA TCG ACC 144 Gin Ser Tyr Val Ser Ser Leu Asn Ser He Arg Thr Glu He Ser Thr 35 40 45 CCT CTT GAA CAT ATA TCT CAG GGG ACC ACA TCG GTG TCT GTT ATT AAC 192 Pro Leu Glu His He Ser Gin Gly Thr Thr Ser Val Ser Val He Asn 50 55 60 CAC ACC CAC GGC AGT TAT TTT GCT GTG GAT ATA CGA GGG CTT GAT GTC 240 His Thr His Gly Ser Tyr Phe Ala Val Asp He Arg Gly Leu Asp Val 65 70 75 80 TAT CAG GCG CGT TTT GAC CAT CTT CGT CTG ATT ATT GAG CAA AAT AAT 288 Tyr Gin Ala Arg Phe Asp His Leu Arg Leu He He Glu Gin Asn Asn 85 90 95 TTA TAT GTG GCA GGG TTC GTT AAT ACG GCA ACA AAT ACT TTC TAC CGT 336 Leu Tyr Val Ala Gly Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg 100 105 110 TTT TCA GAT TTT ACA CAT ATA TCA GTG CCC GGT GTG ACA ACG GTT TCC 384 Phe Ser Asp Phe Thr His He Ser Val Pro Gly Val Thr Thr Val Ser 115 120 125 ATG ACA ACG GAC AGC AGT TAT ACC ACT CTG CAA CGT GTC GCA GCG CTG 432 Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Ala Leu 130 ' 135 140 GAA CGT TCC GGA ATG CAA ATC AGT CGT CAC TCA CTG GTT TCA TCA TAT 480 Glu Arg Ser Gly Met Gin He Ser Arg His Ser Leu Val Ser Ser Tyr 145 150 155 160 CTG GCG TTA ATG GAG TTC AGT GGT AAT ACA ATG ACC AGA GAT GCA TCC 528 Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser 165 170 175 AGA GCA GTT CTG CGT TTT GTC ACT GTC ACA GCA GAA GCC TTA CGC TTC 576 Arg Ala Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe 180 185 190 AGG CAG ATA CAG AGA GAA TTT CGT CAG GCA CTG TCT GAA ACT GCT CCT 624 Arg Gin He Gin Arg Glu Phe Arg Gin Ala Leu Ser Glu Thr Ala Pro 195 200 205 GTG TAT ACG ATG ACG CCG GGA GAC GTG GAC CTC ACT CTG AAC TGG GGG 672 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly 210 215 220 CGA ATC AGC AAT GTG CTT CCG GAG TAT CGG GGA GAG GAT GGT GTC AGA 720 Arg He Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg 225 230 235 240 GTG GGG AGA ATA TCC TTT AAT AAT ATA TCA GCG ATA CTG GGG ACT GTG 768 Val Gly Arg He Ser Phe Asn Asn He Ser Ala He Leu Gly Thr Val 245 250 255 GCC GTT ATA CTG AAT TGC CAT CAT CAG GGG GCG CGT TCT GTT CGC GCC 816 Ala Val He Leu Asn Cys His His Gin Gly Ala Arg Ser Val Arg Ala 260 265 270 GTG AAT GAA GAG AGT CAA CCA GAA TGT CAG ATA ACT GGC GAC AGG CCT 864 Val Asn Glu Glu Ser Gin Pro Glu Cys Gin He Thr Gly Asp Arg Pro 275 280 285 GTT ATA AAA ATA AAC AAT ACA TTA TGG GAA AGT AAT ACA GCT GCA GCG 912 Val He Lys He Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala 290 295 300 TTT CTG AAC AGA AAG TCA CAG TTT TTA TAT ACA ACG GGT AAA CTC GAG 960 Phe Leu Asn Arg Lys Ser Gin Phe Leu Tyr Thr Thr Gly Lys Leu Glu 305 310 315 320 CAC CAC CAC CAC CAC CAC TG 981 325 (2) INFORMATION FOR SEQ ID NO:25: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 326 ammo acids (B) TYPE: ammo acid (D) TOPOLOGY: linear Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Met Lys Cys He Leu Phe Lys Trp Val Leu Cys Leu Leu Leu Gly Phe 1 5 10 15 Ser Ser Val Ser Tyr Ser Arg Glu Phe Thr He Asp Phe Ser Thr Gin 20 25 30 Gin Ser Tyr Val Ser Ser Leu Asn Ser He Arg Thr Glu He Ser Thr 35 40 45 Pro Leu Glu His He Ser Gin Gly Thr Thr Ser Val Ser Val He Asn 50 55 ' 60 His Thr His Gly Ser Tyr Phe Ala Val Asp He Arg Gly Leu Asp Val 65 70 75 80 Tyr Gin Ala Arg Phe Asp His Leu Arg Leu He He Glu Gin Asn Asn 85 90 95 Leu Tyr Val Ala Gly Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg 100 105 110 Phe Ser Asp Phe Thr His He Ser Val Pro Gly Val Thr Thr Val Ser 115 120 125 Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Ala Leu 130 135 140 Glu Arg Ser Gly Met Gin He Ser Arg His Ser Leu Val Ser Ser Tyr 145 150 155 160 Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser 165 170 175 Arg Ala Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe 180 185 190 Arg Gin He Gin Arg Glu Phe Arg Gin Ala Leu Ser Glu Thr Ala Pro 195 200 205 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly 210 215 220 Arg He Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg 225 230 235 240 Val Gly Arg He Ser Phe Asn Asn He Ser Ala He Leu Gly Thr Val 245 250 255 Ala Val He Leu Asn Cys His His Gin Gly Ala Arg Ser Val Arg Ala 260 265 270 Val Asn Glu Glu Ser Gin Pro Glu Cys Gin He Thr Gly Asp Arg Pro 275 280 285 Val He Lys He Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala 290 295 300 Phe Leu Asn Arg Lvs Ser Gin Phe Leu Tyr Thr Thr Gly Lvs Leu Glu 305 " 310 315 320 325 (2) INFORMATION FOR SEQ ID NO:26: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 294 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) Ux) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..294 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATG AAG AAG ATG TTT ATG GCG GTT TTA TTT GCA TTA GCT TCT GTT AAT 48 Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Val Asn 1 * 5 10 15 GCA ATG GCG GCG GAT TGT GCT AAA GGT AAA ATT GAG TTT TCC AAG TAT 96 Ala Met Ala Ala Asp Cys Ala Lys Gly Lys He Glu Phe Ser Lys Tvr 20 25 30 AAT GAG GAT GAC ACA TTT ACA GTG AAG GTT GAC GGG AAA GAA TAC TGG 144 Asn Glu Asp Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp 35 40 45 ACC AGT CGC TGG AAT CTG CAA CCG TTA CTG CAA AGT GCT CAG TTG ACA 192 Thr Ser Arg Trp Asn Leu Gin Pro Leu Leu Gin Ser Ala Gin Leu Thr 50 55 60 GGA ATG ACT GTC ACA ATC AAA TCC AGT ACC TGT GAA TCA GGC TCC GGA 240 Gly Met Thr Val Thr He Lys Ser Ser Thr Cys Glu Ser Gly Ser Giv¬ es 70 75 80 TTT GCT GAA GTG CAG TTT AAT AAT GAC CTC GAG CAC CAC CAC CAC CAC 288 Phe Ala Glu Val Gin Phe Asn Asn Asp Leu Glu His His His His His 85 90 95 CAC TG 294 His (2) INFORMATION FOR SEQ ID NO:27: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 97 ammo acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Val Asn 1 5 10 15 Ala Met Ala Ala Asp Cys Ala Lys Gly Lys He Glu Phe Ser Lys Tyr 20 25 30 Asn Glu Asp Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp 35 40 45 Thr Ser Arσ Trp Asn Leu Gin Pro Leu Leu Gin Ser Ala Gin Leu Thr 50 " 55 60 Gly Met Thr Val Thr He Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly 65 70 75 80 Phe Ala Glu Val Gin Phe Asn Asn Asp Leu Glu His His His His His 85 90 95 His (2) INFORMATION FOR SEQ ID NO:28: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs !B) TYPE: nucleic acid :C) STRANDEDNESS: single ;D' TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:28: CGGAATTCAA GGAATTTACC TTAGACTTCT CG 32 (2) INFORMATION FOR SEQ ID NO:29: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGCTCGAGTC AACTGCTAAT AGTTCTGC 28 (2) INFORMATION FOR SEQ ID NO:30: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: CGGAATTCCG GGAGTTTACG ATAGACTTTT CG 32 (2) INFORMATION FOR SEQ ID NO:31: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31. GGCTCGAGTT ATTTACCCGT TGTATATAA 29 (2) INFORMATION FOR SEQ ID NO: 32: ' l) SEQUENCE CHARACTERISTICS : (A) LENGTH: 2127 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear m) MOLECULE TYPE: DNA (genomic) Ux) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..2127 I I) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ATG AAA ATA AAA ACA GGT GCA CGC ATC CTC GCA TTA TCC GCA TTA ACG 48 Met Lys He Lys Thr Gly Ala Arg He Leu Ala Leu Ser Ala Leu Tnr 1 * 5 10 15 ACG ATG ATG TTT TCC GCC TCG GCT CTC GCC AAA ATC GAA GAA GGT AAA 96 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys He Glu Glu Gly Lys 20 25 30 CTG GTA ATC TGG ATT AAC GGC GAT AAA GGC TAT AAC GGT CTC GCT GAA 144 Leu Val He Trp He Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 GTC C-GT AAG AAA TTC GAG AAA GAT ACC GGA ATT AAA GTC ACC GTT GAG 192 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly He Lys Val Thr Val Glu 50 55 60 CAT CCG GAT AAA CTG GAA GAG AAA TTC CCA CAG GTT GCG GCA ACT GGC 240 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gin Val Ala Ala Thr Gly 65 70 75 80 GAT GGC CCT GAC ATT ATC TTC TGG GCA CAC GAC CGC TTT GGT GGC TAC 286 Asp Gly Pro Asp He He Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 GCT CAA TCT GGC CTG TTG GCT GAA ATC ACC CCG GAC AAA GCG TTC CAG 336 Ala Gin Ser Gly Leu Leu Ala Glu He Thr Pro Asp Lys Ala Phe Gin 100 105 110 GAC AAG CTG TAT CCG TTT ACC TGG GAT GCC GTA CGT TAC AAC GGC AAG 384 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 CTG ATT GCT TAC CCG ATC GCT GTT GAA GCG TTA TCG CTG ATT TAT AAC 432 Leu He Ala Tyr Pro He Ala Val Glu Ala Leu Ser Leu He Tyr Asn 130 135 140 AAA GAT CTG CTG CCG AAC CCG CCA AAA ACC TGG GAA GAG ATC CCG GCG 480 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu He Pro Ala 145 150 155 160 CTG GAT AAA GAA CTG AAA GCG AAA GGT AAG AGC GCG CTG ATG TTC AAC 528 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 CTG CAA GAA CCG TAC TTC ACC TGG CCG CTG ATT GCT GCT GAC GGG GGT 576 Leu Gin Glu Pro Tyr Phe Thr Trp Pro Leu He Ala Ala Asp Gly Gly 180 185 190 TAT GCG TTC AAG TAT GAA AAC GGC AAG TAC GAC ATT AAA GAC GTG GGC 624 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp He Lys Asp Val Gly 195 200 205 GTG GAT AAC GCT GGC GCG AAA GCG GGT CTG ACC TTC CTG GTT GAC CTG 672 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 ATT AAA AAC AAA CAC ATG AAT GCA GAC ACC GAT TAC TCC ATC GCA GAA 720 He Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser He Ala Glu 225 230 235 240 GCT GCC TTT AAT AAA GGC GAA ACA GCG ATG ACC ATC AAC GGC CCG TGG 768 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr He Asn Gly Pro Trp 245 250 255 GCA TGG TCC AAC ATC GAC ACC AGC AAA GTG AAT TAT GGT GTA ACG GTA 816 Ala Trp Ser Asn He Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 CTG CCG ACC TTC AAG GGT CAA CCA TCC AAA CCG TTC GTT GGC GTG CTG 864 Leu Pro Thr Phe Lys Gly Gin Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 AGC GCA GGT ATT AAC GCC GCC AGT CCG AAC AAA GAG CTG GCG AAA GAG 912 Ser Ala Gly He Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 TTC CTC GAA AAC TAT CTG CTG ACT GAT GAA GGT CTG GAA GCG GTT AAT 960 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 ' 310 315 320 AAA GAC AAA CCG CTG GGT GCC GTA GCG CTG AAG TCT TAC GAG GAA GAG 1008 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tvr Glu Glu Glu 325 330 ' 335 TTG GCG AAA GAT CCA CGT ATT GCC GCC ACC ATG GAA AAC GCC CAG AAA 1056 Leu Ala Lys Asp Pro Arg He Ala Ala Thr Met Glu Asn Ala Gin Lys 340 345 350 GGT GAA ATC ATG CCG AAC ATC CCG CAG ATG TCC GCT TTC TGG TAT GCC 1104 Gly Glu He Met Pro Asn He Pro Gin Met Ser Ala Phe Trp Tyr Ala 355 360 365 GTG CGT ACT GCG GTG ATC AAC GCC GCC AGC GGT CGT CAG ACT GTC GAT 1152 Val Arg Thr Ala Val He Asn Ala Ala Ser Gly Arg Gin Thr Val Asp 370 375 380 GAA GCC CTG AAA GAC GCG CAG ACT TCG AGC TCG AAC AAC AAC AAC AAT 1200 Glu Ala Leu Lys Asp Ala Gin Thr Ser Ser Ser Asn Asn Asn Asn Asn 385 390 395 400 AAC AAT AAC AAC AAC CTC GGG ATC GAG GGA AGG ATT TCA GAA TTC AAG 1248 Asn Asn Asn Asn Asn Leu Gly He Glu Gly Arg He Ser Glu Phe Lys 405 410 415 GAA TTT ACC TTA GAC TTC TCG ACT GCA AAG ACG TAT GTA GAT TCG CTG 1296 Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser Leu 420 425 430 AAT GTC ATT CGC TCT GCA ATA GGT ACT CCA TTA CAG ACT ATT TCA TCA 1344 Asn Val He Arg Ser Ala He Gly Thr Pro Leu Gin Thr He Ser Ser 435 440 445 GGA GGT ACG TCT TTA CTG ATG ATT GAT AGT GGC TCA GGG GAT AAT TTG 1392 Gly Gly Thr Ser Leu Leu Met He Asp Ser Gly Ser Gly Asp Asn Leu 450 455 460 TTT GCA GTT GAT GTC AGA GGG ATA GAT GCA GAG GAA GGG CGG TTT AAT 1440 Phe Ala Val Asp Val Arg Gly He Asp Ala Glu Glu Gly Arg Phe Asn 465 470 475 480 AAT CTA CGG CTT ATT GTT GAA CGA AAT AAT TTA TAT GTG ACA GGA TTT 1488 Asn Leu Arg Leu He Val Glu Arg Asn Asn Leu Tyr Val Thr Gly Phe 485 490 495 GTT AAC AGG ACA AAT AAT GTT TTT TAT CGC TTT GCT GAT TTT TCA CAT 1536 Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser His 500 505 510 GTT ACC TTT CCA GGT ACA ACA GCG GTT ACA TTG TCT GGT GAC AGT AGC 1584 Val Thr Phe Pro Glv Thr Thr Ala Val Thr Leu Ser Gly Asp Ser Ser 515 520 525 TAT ACC ACG TTA CAG CGT GTT GCA GGG ATC AGT CGT ACG GGG ATG CAG 1632 Tyr Thr Thr Leu Gin Arg Val Ala Gly He Ser Arg Thr Gly Met Gin 530 535 540 ATA AAT CGC CAT TCG TTG ACT ACT TCT TAT CTG GAT TTA ATG TCG CAT 1680 He Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser His 545 550 555 560 AGT GGA ACC TCA CTG ACG CAG TCT GTG GCA AGA GCG ATG TTA CGG TTT 1728 Ser Gly Thr Ser Leu Thr Gin Ser Val Ala Arg Ala Met Leu Arg Phe 565 570 575 GTT ACT GTG ACA GCT GAA GCT TTA CGT TTT CGG CAA ATA CAG AGG GGA 1776 Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He Gin Arg Gly 580 585 590 TTT CGT ACA ACA CTG GAT GAT CTC AGT GGG CGT TCT TAT GTA ATG ACT 1824 Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met Thr 595 600 605 GCT GAA GAT GTT GAT CTT ACA TTG AAC TGG GGA AGG TTG AGT AGC GTC 1872 Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser Val 610 615 620 CTG CCT GAC TAT CAT GGA CAA GAC TCT GTT CGT GTA GGA AGA ATT TCT 1920 Leu Pro Asp Tyr His Gly Gin Asp Ser Val Arg Val Gly Arg He Ser 625 630 635 640 TTT GGA AGC ATT AAT GCA ATT CTG GGA AGC GTG GCA TTA ATA CTG AAT 1968 Phe Gly Ser He Asn Ala He Leu Gly Ser Val Ala Leu He Leu Asn 645 650 655 TGT CAT CAT CAT GCA TCG CGA GTT GCC AGA ATG GCA TCT GAT GAG TTT 2016 Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu Phe 660 665 670 CCT TCT ATG TGT CCG GCA GAT GGA AGA GTC CGT GGG ATT ACG CAC AAT 2064 Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly He Thr His Asn 675 680 685 AAA ATA TTG TGG GAT TCA TCC ACT CTG GGG GCA ATT CTG ATG CGC AGA 2112 Lys He Leu Trp Asp Ser Ser Thr Leu Gly Ala He Leu Met Arg Arg 690 695 700 ACT ATT AGC AGT TG 2127 Thr He Ser Ser 705 (2) INFORMATION FOR SEQ ID NO:33: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 708 ammo acids (B) TYPE: ammo acid (D) TOPOLOGY, linear Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33 Met Lys He Lys Thr Gly Ala Arg He Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Th: Met Met Phe Ser Ala Ser Ala Leu Ala Lys He Glu Glu Gly Lys 20 25 30 Leu Val He Trp He Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly He Lys Val Thr Val Glu 50 * 55 60 His Pro Asp Lvs Leu Glu Glu Lys Phe Pro Gin Val Ala Ala Thr Gly 65 ' 70 75 80 Asp Gly Pro Asp He He Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gin Ser Gly Leu Leu Ala Glu He Thr Pro Asp Lys Ala Phe Gin 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu He Ala Tyr Pro He Ala Val Glu Ala Leu Ser Leu He Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu He Pro Ala 145 150 ' 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gin Glu Pro Tyr Phe Thr Trp Pro Leu He Ala Ala Asp Gly Gly 180 165 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp He Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 ' 220 He Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser He Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr He Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn He Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gin Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly He Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg He Ala Ala Thr Met Glu Asn Ala Gin Lys 340 345 350 Gly Glu He Met Pro Asn He Pro Gin Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Arg Thr Ala Val He Asn Ala Ala Ser Gly Arg Gin Thr Val Asp 370 375 380 Glu Ala Leu Lys Asp Ala Gin Thr Ser Ser Ser Asn Asn Asn Asn Asn 385 390 395 400 Asn Asn Asn Asn Asn Leu Gly He Glu Gly Arg He Ser Glu Phe Lys 405 410 415 Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser Leu 420 425 430 Asn Val He Arg Ser Ala He Gly Thr Pro Leu Gin Thr He Ser Ser 435 440 445 Gly Gly Thr Ser Leu Leu Met He Asp Ser Gly Ser Gly Asp Asn Leu 450 455 460 Phe Ala Val Asp Val Arg Gly He Asp Ala Glu Glu Gly Arg Phe Asn 465 470 " 475 480 Asn Leu Arg Leu He Val Glu Arg Asn Asn Leu Tyr Val Thr Gly Phe 485 490 495 Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser His 500 505 510 Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser Ser 515 520 525 Tyr Thr Thr Leu Gin Arg Val Ala Gly He Ser Arg Thr Gly Met Gin 530 535 540 He Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser His 545 550 555 560 Ser Gly Thr Ser Leu Thr Gin Ser Val Ala Arg Ala Met Leu Arg Phe 565 570 575 Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He Gin Arg Gly 580 585 590 Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met Thr 595 600 605 Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser Val 610 615 620 Leu Pro Asp Tyr His Gly Gin Asp Ser Val Arg Val Glv Arg He Ser 625 630 635 * 640 Phe Gly Ser He Asn Ala He Leu Gly Ser Val Ala Leu He Leu Asn 645 650 655 Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu Phe 660 665 670 Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly He Thr His Asn 675 680 685 Lys He Leu Trp Asp Ser Ser Thr Leu Gly Ala He Leu Met Arg Arg 690 695 700 Thr He Ser Ser 705 (2) INFORMATION FOR SEQ ID NO:34: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2136 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) Ux) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..2136 (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:34: ATG AAA ATA AAA ACA GGT GCA CGC ATC CTC GCA TTA TCC GCA TTA ACG 48 Met Lys He Lys Thr Gly Ala Arg He Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 ACG ATG ATG TTT TCC GCC TCG GCT CTC GCC AAA ATC GAA GAA GGT AAA 96 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys He Glu Glu Gly Lys 20 25 30 CTG GTA ATC TGG ATT AAC GGC GAT AAA GGC TAT AAC GGT CTC GCT GAA 144 Leu Val He Trp He Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 GTC GGT AAG AAA TTC GAG AAA GAT ACC GGA ATT AAA GTC ACC GTT GAG 192 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly He Lys Val Thr Val Glu 50 55 60 CAT CCG GAT AAA CTG GAA GAG AAA TTC CCA CAG GTT GCG GCA ACT GGC 240 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gin Val Ala Ala Thr Gly 65 70 75 80 GAT GGC CCT GAC ATT ATC TTC TGG GCA CAC GAC CGC TTT GGT GGC TAC 288 Asp Gly Pro Asp He He Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 GCT CAA TCT GGC CTG TTG GCT GAA ATC ACC CCG GAC AAA GCG TTC CAG 336 Ala Gin Ser Gly Leu Leu Ala Glu He Thr Pro Asp Lys Ala Phe Gin 100 105 " 110 GAC AAG CTG TAT CCG TTT ACC TGG GAT GCC GTA CGT TAC AAC GGC AAG 384 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 CTG ATT GCT TAC CCG ATC GCT GTT GAA GCG TTA TCG CTG ATT TAT AAC 432 Leu He Ala Tyr Pro He Ala Val Glu Ala Leu Ser Leu He Tyr Asn 130 135 140 AAA GAT CTG CTG CCG AAC CCG CCA AAA ACC TGG GAA GAG ATC CCG GCG 480 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu He Pro Ala 145 150 155 160 CTG GAT AAA GAA CTG AAA GCG AAA GGT AAG AGC GCG CTG ATG TTC AAC 528 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 CTG CAA GAA CCG TAC TTC ACC TGG CCG CTG ATT GCT GCT GAC GGG GGT 576 Leu Gin Glu Pro Tvr Phe Thr Trp Pro Leu He Ala Ala Asp Gly Gly 180 " 185 190 TAT GCG TTC AAG TAT GAA AAC GGC AAG TAC GAC ATT AAA GAC GTG GGC 624 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp He Lys Asp Val Gly 195 ' 200 205 GTG GAT AAC GCT GGC GCG AAA GCG GGT CTG ACC TTC CTG GTT GAC CTG 672 Val Asp Asn Ala Glv Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 ' 215 220 ATT AAA AAC AAA CAC ATG AAT GCA GAC ACC GAT TAC TCC ATC GCA GAA 720 He Lys Asn Lys H s Met Asn Ala Asp Thr Asp Tyr Ser He Ala Glu 225 230 235 240 GCT GCC TTT AAT AAA GGC GAA ACA GCG ATG ACC ATC AAC GGC CCG TGG 768 Ala Ala Phe Asn Lvs Gly Glu Thr Ala Met Thr He Asn Gly Pro Trp 245 250 255 GCA TGG TCC AAC ATC GAC ACC AGC AAA GTG AAT TAT GGT GTA ACG GTA 816 Ala Trp Ser Asn lie Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 CTG CCG ACC TTC AAG GGT CAA CCA TCC AAA CCG TTC GTT GGC GTG CTG 864 Leu Pro Thr Phe Lvs Gly Gin Pro Ser Lys Pro Phe Val Gly Val Leu 275 ' ' 280 285 AGC GCA GGT ATT AAC GCC GCC AGT CCG AAC AAA GAG CTG GCG AAA GAG 912 Ser Ala Gly He Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 TTC CTC GAA AAC TAT CTG CTG ACT GAT GAA GGT CTG GAA GCG GTT AAT 960 Phe Leu Glu Asn Tvr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 " 310 315 320 AAA GAC AAA CCG CTG GGT GCC GTA GCG CTG AAG TCT TAC GAG GAA GAG 1008 Lys Asp Lys Pro Leu Glv Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 ' 330 335 TTG GCG AAA GAT CCA CGT ATT GCC GCC ACC ATG GAA AAC GCC CAG AAA 1056 Leu Ala Lys Asp Pro Arg He Ala Ala Thr Met Glu Asn Ala Gin Lys 340 345 350 GGT GAA ATC ATG CCG AAC ATC CCG CAG ATG TCC GCT TTC TGG TAT GCC 1104 Gly Glu He Met Pro Asn He Pro Gin Met Ser Ala Phe Trp Tyr Ala 355 360 365 GTG CGT ACT GCG GTG ATC AAC GCC GCC AGC GGT CGT CAG ACT GTC GAT 1152 Val Arg Thr Ala Val He Asn Ala Ala Ser Gly Arg Gin Thr Val Asp 370 375 380 GAA GCC CTG AAA GAC GCG CAG ACT TCG AGC TCG AAC AAC AAC AAC AAT 1200 Glu Ala Leu Lys Asp Ala Gin Thr Ser Ser Ser Asn Asn Asn Asn Asn 385 390 395 400 AAC AAT AAC AAC AAC CTC GGG ATC GAG GGA AGG ATT TCA GAA TTC CGG 1248 Asn Asn Asn Asn Asn Leu Gly He Glu Gly Arg He Ser Glu Phe Arg 405 410 415 GAG TTT ACG ATA GAC TTT TCG ACC CAA CAA AGT TAT GTC TCT TCG TTA 1296 Glu Phe Thr He Asp Phe Ser Thr Gin Gin Ser Tyr Val Ser Ser Leu 420 425 430 AAT AGT ATA CGG ACA GAG ATA TCG ACC CCT CTT GAA CAT ATA TCT CAG 1344 Asn Ser He Arg Thr Glu He Ser Thr Pro Leu Glu His He Ser Gin 435 440 445 GGG ACC ACA TCG GTG TCT GTT ATT AAC CAC ACC CAC GGC AGT TAT TTT 1392 Gly Thr Thr Ser Val Ser Val He Asn His Thr His Gly Ser Tyr Phe 450 455 460 GCT GTG GAT ATA CGA GGG CTT GAT GTC TAT CAG GCG CGT TTT GAC CAT 1440 Ala Val Asp He Arg Gly Leu Asp Val Tyr Gin Ala Arg Phe Asp His 465 * 470 475 " 480 CTT CGT CTG ATT ATT GAG CAA AAT AAT TTA TAT GTG GCA GGG TTC GTT 1488 Leu Arg Leu He He Glu Gin Asn Asn Leu Tyr Val Ala Gly Phe Val 485 490 495 AAT ACG GCA ACA AAT ACT TTC TAC CGT TTT TCA GAT TTT ACA CAT ATA 1536 Asn Thr Ala Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr His He 500 505 510 TCA GTG CCC GGT GTG ACA ACG GTT TCC ATG ACA ACG GAC AGC AGT TAT 1584 Ser Val Pro Gly Val Thr Thr Val Ser Met Thr Thr Asp Ser Ser Tyr 515 520 525 ACC ACT CTG CAA CGT GTC GCA GCG CTG GAA CGT TCC GGA ATG CAA ATC 1632 Thr Thr Leu Gin Arg Val Ala Ala Leu Glu Arg Ser Gly Met Gin He 530 535 540 AGT CGT CAC TCA CTG GTT TCA TCA TAT CTG GCG TTA ATG GAG TTC AGT 1680 Ser Arg H s Ser Leu Val Ser Ser Tyr Leu Ala Leu Met Glu Phe Ser 545 550 555 560 GGT AAT ACA ATG ACC AGA GAT GCA TCC AGA GCA GTT CTG CGT TTT GTC 1728 Gly Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg Phe Val 565 570 575 ACT GTC ACA GCA GAA GCC TTA CGC TTC AGG CAG ATA CAG AGA GAA TTT 1776 Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He Gin Arg Glu Phe 580 585 590 CGT CAG GCA CTG TCT GAA ACT GCT CCT GTG TAT ACG ATG ACG CCG GGA 1824 Arg Gin Ala Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr Pro Gly 595 600 605 GAC GTG GAC CTC ACT CTG AAC TGG GGG CGA ATC AGC AAT GTG CTT CCG 1872 Asp Val As:: Leu Thr Leu Asn Trp Gly Arg He Ser Asn Val Leu Pro 610 " 615 620 GAG TAT CGG GGA GAG GAT GGT GTC AGA GTG GGG AGA ATA TCC TTT AAT 1920 Glu Tyr Arg Gly Glu Asp Gly Val Arg Val Gly Arg He Ser Phe Asn 625 630 635 640 AAT ATA TCA GCG ATA CTG GGG ACT GTG GCC GTT ATA CTG AAT TGC CAT 1966 Asn He Ser Ala He Leu Gly Thr Val Ala Val He Leu Asn Cys His 645 650 655 CAT CAG GGG GCG CGT TCT GTT CGC GCC GTG AAT GAA GAG AGT CAA CCA 2016 His Gin Gly Ala Arg Ser Val Arg Ala Val Asn Glu Glu Ser Gin Pro 660 665 670 GAA TGT CAG ATA ACT GGC GAC AGG CCT GTT ATA AAA ATA AAC AAT ACA 2064 Glu Cys Gin He Thr Gly Asp Arg Pro Val He Lys He Asn Asn Thr 675 680 685 TTA TGG GAA AGT AAT ACA GCT GCA GCG TTT CTG AAC AGA AAG TCA CAG 2112 Leu Trp Glu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys Ser Gin 690 695 700 TTT TTA TAT ACA ACG GGT AAA TA 2136 Phe Leu Tyr Thr Thr Gly Lys 705 710 (2) INFORMATION FOR SEQ ID NO:35: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 711 ammo acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Met Lys He Lys Thr Gly Ala Arg He Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys He Glu Glu Gly Lys 20 25 30 Leu Val He Trp He Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 ' 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly He Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gin Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp He He Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gin Ser Gly Leu Leu Ala Glu He Thr Pro Asp Lys Ala Phe Gin 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu He Ala Tyr Pro He Ala Val Glu Ala Leu Ser Leu He Tyr Asn 130 ' 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu He Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Glv Lys Ser Ala Leu Met Phe Asn 165 ' 170 175 Leu Gin Glu Pro Tyr Phe Thr Trp Pro Leu He Ala Ala Asp Gly Gly 180 ' 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp He Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 He Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser He Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr He Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn He Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gin Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly He Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg He Ala Ala Thr Met Glu Asn Ala Gin Lys 340 345 350 Gly Glu He Met Pro Asn He Pro Gin Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Arg Thr Ala Val He Asn Ala Ala Ser Gly Arg Gin Thr Val Asp 370 375 380 Glu Ala Leu Lys Asp Ala Gin Thr Ser Ser Ser Asn Asn Asn Asn Asn 385 390 395 400 Asn Asn Asn Asn Asn Leu Gly He Glu Gly Arg He Ser Glu Phe Arg 405 410 415 Glu Phe Thr He Asp Phe Ser Thr Gin Gin Ser Tyr Val Ser Ser Leu 420 425 430 Asn Ser He Arg Thr Glu He Ser Thr Pro Leu Glu His He Ser Gin 435 440 445 Gly Thr Thr Ser Val Ser Val He Asn His Thr His Gly Ser Tyr Phe 450 455 460 Ala Val Asp He Arg Gly Leu Asp Val Tyr Gin Ala Arg Phe Asp His 465 470 475 480 Leu Arg Leu He He Glu Gin Asn Asn Leu Tyr Val Ala Gly Phe Val 485 490 495 Asn Thr Ala Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr His He 500 505 510 Ser Val Pro Gly Val Thr Thr Val Ser Met Thr Thr Asp Ser Ser Tyr 515 520 525 Thr Thr Leu Gin Arg Val Ala Ala Leu Glu Arg Ser Gly Met Gin He 530 535 540 Ser Arg His Ser Leu Val Ser Ser Tyr Leu Ala Leu Met Glu Phe Ser 545 550 555 560 Gly Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg Phe Val 565 570 575 Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He Gin Arg Glu Phe 580 585 590 Arg Gin Ala Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr Pro Glv 595 600 605 Asp Val Asp Leu Thr Leu Asn Trp Gly Arg He Ser Asn Val Leu Pro 610 615 620 Glu Tyr Arg Gly Glu Asp Gly Val Arg Val Gly Arg He Ser Phe Asn 625 630 635 640 Asn He Ser Ala He Leu Gly Thr Val Ala Val He Leu Asn Cys His 645 650 655 His Gin Gly Ala Arg Ser Val Arg Ala Val Asn Glu Glu Ser Gin Pro 660 665 670 Glu Cys Gin He Thr Gly Asp Arg Pro Val He Lys He Asn Asn Thr 675 ' 680 685 Leu Trp Glu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys Ser Gin 690 695 700 Phe Leu Tyr Thr Thr Gly Lys 705 710 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 981 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..981 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT 48 Met Lys Lys Thr Ala He Ala He Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 ACC GTT GCG CAA GCT GAC TAC AAG GAC GAC GAT GAC AAG AAG CTT GAA 96 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Glu 20 25 30 TTC AAG GAA TTT ACC TTA GAC TTC TCG ACT GCA AAG ACG TAT GTA GAT 144 Phe Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 35 40 45 TCG CTG AAT GTC ATT CGC TCT GCA ATA GGT ACT CCA TTA CAG ACT ATT 192 Ser Leu Asn Val He Arg Ser Ala He Gly Thr Pro Leu Gin Thr He 50 " 55 60 TCA TCA GGA GGT ACG TCT TTA CTG ATG ATT GAT AGT GGC TCA GGG GAT 240 Ser Ser Gly Gly Thr Ser Leu Leu Met He Asp Ser Gly Ser Gly Asp 65 70 75 ' 80 AAT TTG TTT GCA GTT GAT GTC AGA GGG ATA GAT GCA GAG GAA GGG CGG 288 Asn Leu Phe Ala Val Aso Val Arg Gly He Asp Ala Glu Glu Gly Arg 85 " 90 95 TTT AAT AAT CTA CGG CTT ATT GTT GAA CGA AAT AAT TTA TAT GTG ACA 336 Phe Asn Asn Leu Arg Leu He Val Glu Arg Asn Asn Leu Tyr Val Thr 100 105 110 GGA TTT GTT AAC AGG ACA AAT AAT GTT TTT TAT CGC TTT GCT GAT TTT 384 Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 115 120 125 TCA CAT GTT ACC TTT CCA GGT ACA ACA GCG GTT ACA TTG TCT GGT GAC 432 Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asu 130 135 140 AGT AGC TAT ACC ACG TTA CAG CGT GTT GCA GGG ATC AGT CGT ACG GGG 480 Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Gly He Ser Arg Thr Glv 145 150 155 16 * 0 ATG CAG ATA AAT CGC CAT TCG TTG ACT ACT TCT TAT CTG GAT TTA ATG 528 Met Gin He Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 165 170 175 TCG CAT AGT GGA ACC TCA CTG ACG CAG TCT GTG GCA AGA GCG ATG TTA 576 Ser His Ser Gly Thr Ser Leu Thr Gin Ser Val Ala Arg Ala Met Leu 180 185 190 CGG TTT GTT ACT GTG ACA GCT GAA GCT TTA CGT TTT CGG CAA ATA CAG 624 Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He Gin 195 200 205 AGG GGA TTT CGT ACA ACA CTG GAT GAT CTC AGT GGG CGT TCT TAT GTA 672 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val 210 215 220 ATG ACT GCT GAA GAT GTT GAT CTT ACA TTG AAC TGG GGA AGG TTG AGT 720 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 225 230 235 240 AGC GTC CTG CCT GAC TAT CAT GGA CAA GAC TCT GTT CGT GTA GGA AGA 766 Ser Val Leu Pro Asp Tyr His Gly Gin Asp Ser Val Arg Val Gly Arg 245 250 255 ATT TCT TTT GGA AGC ATT AAT GCA ATT CTG GGA AGC GTG GCA TTA ATA 816 He Ser Phe Gly Ser He Asn Ala He Leu Gly Ser Val Ala Leu He 260 265 * 270 CTG AAT TGT CAT CAT CAT GCA TCG CGA GTT GCC AGA ATG GCA TCT GAT 864 Leu Asn Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp 275 280 285 GAG TTT CCT TCT ATG TGT CCG GCA GAT GGA AGA GTC CGT GGG ATT ACG 912 Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly He Thr 290 295 300 CAC AAT AAA ATA TTG TGG GAT TCA TCC ACT CTG GGG GCA ATT CTG ATG 960 His Asn Lys He Leu Trp Asp Ser Ser Thr Leu Gly Ala He Leu Met 305 310 315 320 CGC AGA ACT ATT AGC AGT TG 981 Arg Arg Thr He Ser Ser 325 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 326 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37 : Met Lys Lys Thr Ala He Ala He Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Glu 20 25 30 Phe Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 35 40 45 Ser Leu Asn Val He Arg Ser Ala He Gly Thr Pro Leu Gin Thr He 50 55 60 Ser Ser Gly Gly Thr Ser Leu Leu Met He Asp Ser Gly Ser Gly Asp 65 70 75 80 Asn Leu Phe Ala Val Asp Val Arg Gly He Asp Ala Glu Glu Gly Arg 85 90 95 Phe Asn Asn Leu Arg Leu He Val Glu Arg Asn Asn Leu Tyr Val Thr 100 105 110 Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 115 120 125 Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp 130 135 140 Ser Ser Tyr Thr Thr Leu Gin Arg Val Ala Gly He Ser Arg Thr Gly 145 150 155 16 * 0 Met Gin He Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 165 170 175 Ser His Ser Gly Thr Ser Leu Thr Gin Ser Val Ala Arg Ala Met Leu 180 165 190 Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He Gin 195 200 205 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val 210 215 220 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 225 230 235 240 Ser Val Leu Pro Asp Tyr His Gly Gin Asp Ser Val Arg Val Gly Arg 245 250 255 He Ser Phe Gly Ser He Asn Ala He Leu Gly Ser Val Ala Leu He 260 265 270 Leu Asn Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp 275 280 285 Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly He Thr 290 295 300 His Asn Lvs He Leu Trp Asp Ser Ser Thr Leu Gly Ala He Leu Met 305 ' 310 315 320 Arg Arg Thr He Ser Ser 325 (2; INFORMATION FOR SEQ ID NO:38: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 990 base pairs (B) TYPE: nucleic acid !C) STRANDEDNESS: single 'D) TOPOLOGY: linear ( ii ) MOLECULE TYPE : DNA (genomic) ( ix ) FEATURE : (A) NAME/KEY: CDS (B) LOCATION: 1..990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT 48 Met Lys Lys Thr Ala He Ala He Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 ACC GTT GCG CAA GCT GAC TAC AAG GAC GAC GAT GAC AAG AAG CTT GAA 96 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Glu 20 25 30 TTC CGG GAG TTT ACG ATA GAC TTT TCG ACC CAA CAA AGT TAT GTC TCT 144 Phe Arg Glu Phe Thr He Asp Phe Ser Thr Gin Gin Ser Tyr Val Ser 35 40 45 TCG TTA AAT AGT ATA CGG ACA GAG ATA TCG ACC CCT CTT GAA CAT ATA 192 Ser Leu Asn Ser He Arg Thr Glu He Ser Thr Pro Leu Glu His He 50 55 60 TCT CAG GGG ACC ACA TCG GTG TCT GTT ATT AAC CAC ACC CAC GGC AGT 240 Ser Gin Gly Thr Thr Ser Val Ser Val He Asn His Thr His Gly Ser 65 70 75 80 TAT TTT GCT GTG GAT ATA CGA GGG CTT GAT GTC TAT CAG GCG CGT TTT 288 Tyr Phe Ala Val Asp He Arg Gly Leu Asp Val Tyr Gin Ala Arg Phe 85 90 95 GAC CAT CTT CGT CTG ATT ATT GAG CAA AAT AAT TTA TAT GTG GCA GGG 336 Asp His Leu Arg Leu He He Glu Gin Asn Asn Leu Tyr Val Ala Gly 100 105 110 TTC GTT AAT ACG GCA ACA AAT ACT TTC TAC CGT TTT TCA GAT TTT ACA 384 Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr 115 120 125 CAT ATA TCA GTG CCC GGT GTG ACA ACG GTT TCC ATG ACA ACG GAC AGC 432 His He Ser Val Pro Gly Val Thr Thr Val Ser Met Thr Thr Asp Ser 130 135 140 AGT TAT ACC ACT CTG CAA CGT GTC GCA GCG CTG GAA CGT TCC GGA ATG 480 Ser Tyr Thr Thr Leu Gin Arg Val Ala Ala Leu Glu Arg Ser Gly Met 145 150 155 160 CAA ATC AGT CGT CAC TCA CTG GTT TCA TCA TAT CTG GCG TTA ATG GAG 528 Gin He Ser Arg His Ser Leu Val Ser Ser Tyr Leu Ala Leu Met Glu 165 170 175 TTC AGT GGT AAT ACA ATG ACC AGA GAT GCA TCC AGA GCA GTT CTG CGT 576 Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg 180 185 190 TTT GTC ACT GTC ACA GCA GAA GCC TTA CGC TTC AGG CAG ATA CAG AGA 624 Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He Gin Arg 195 200 205 GAA TTT CGT CAG GCA CTG TCT GAA ACT GCT CCT GTG TAT ACG ATG ACG 672 Glu Phe Arg Gin Ala Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr 210 215 220 CCG GGA GAC GTG GAC CTC ACT CTG AAC TGG GGG CGA ATC AGC AAT GTG 720 Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly Arg He Ser Asn Val 225 230 235 240 CTT CCG GAG TAT CGG GGA GAG GAT GGT GTC AGA GTG GGG AGA ATA TCC 768 Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg Val Gly Arg He Ser 245 250 255 TTT AAT AAT ATA TCA GCG ATA CTG GGG ACT GTG GCC GTT ATA CTG AAT 816 Phe Asn Asn He Ser Ala He Leu Gly Thr Val Ala Val He Leu Asn 260 265 270 TGC CAT CAT CAG GGG GCG CGT TCT GTT CGC GCC GTG AAT GAA GAG AGT 864 Cys His His Gin Gly Ala Arg Ser Val Arg Ala Val Asn Glu Glu Ser 275 280 285 CAA CCA GAA TGT CAG ATA ACT GGC GAC AGG CCT GTT ATA AAA ATA AAC 912 Gin Pro Glu Cys Gin He Thr Gly Asp Arg Pro Val He Lys He Asn 290 295 300 AAT ACA TTA TGG GAA AGT AAT ACA GCT GCA GCG TTT CTG AAC AGA AAG 960 Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys 305 310 315 320 TCA CAG TTT TTA TAT ACA ACG GGT AAA TA 990 Ser Gin Phe Leu Tyr Thr Thr Gly Lys 325 330 (2) INFORMATION FOR SEQ ID NO:39: U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 329 ammo acids (B! TYPE: amino acid (D) TOPOLOGY: linear Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Met Lys Lys Thr Ala He Ala He Ala Val Ala Leu Ala Glv Phe Ala 1 5 10 15 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Glu 20 " 25 30 Phe Arg Glu Phe Thr He Asp Phe Ser Thr Gin Gin Ser Tyr Val Ser 35 40 45 Ser Leu Asn Ser He Arg Thr Glu He Ser Thr Pro Leu Glu His He 50 55 60 Ser Gin Gly Thr Thr Ser Val Ser Val He Asn His Thr H s Glv Ser 65 70 75 6C Tyr Phe Ala Val Asp He Arg Gly Leu Asp Val Tyr Gin Ala Arg Phe 85 90 95 Asp His Leu Arg Leu He He Glu Gin Asn Asn Leu Tyr Val Ala Gly 100 105 110 Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr 115 120 125 His He Ser Val Pro Gly Val Thr Thr Val Ser Met Thr Thr Asp Ser 130 * 135 140 Ser Tyr Thr Thr Leu Gin Arg Val Ala Ala Leu Glu Arg Ser Gly Met 145 150 155 160 Gin He Ser Arg His Ser Leu Val Ser Ser Tyr Leu Ala Leu Met Glu 165 170 175 Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg 180 185 190 Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gin He G n Arg 195 200 205 Glu Phe Arg Gin Ala Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr 210 215 220 Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly Arg He Ser Asn Val 225 230 235 240 Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg Val Gly Arg He Ser 245 250 255 Phe Asn Asn He Ser Ala He Leu Gly Thr Val Ala Val He Leu Asn 260 265 270 Cys His His Gin Gly Ala Arg Ser Val Arg Ala Val Asn Glu Glu Ser 275 280 285 Gin Pro Glu Cys Gin He Thr Gly Asp Arg Pro Val He Lys He Asn 290 295 300 Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys 305 310 315 320 Ser Gin Phe Leu Tyr Thr Thr Gly Lys 325

Claims: CLAIMS What is claimed is 1 A method of treatment comprising a) providing 0 antitoxin directed against at least a portion ot an Escher ichia coli verotoxin in an aqueous solution in therapeutic amount that is administrable. and n) an intoxicated subject, and b) administering said antitoxin to said subject 2 The method of Claim 1 wherein said Escherichia colt verotoxin is recombinant 3 The method of Claim 1 wherein said antitoxin is an av ian antitoxin 4 The method of Claim 2 wherein said recombinant Escherichia coli v erotoxin is a fusion protein comprising a non- verotoxin protein sequence and a portion of the Eschei ichia coli v erotoxin VTl sequence 5 The method of Claim 2 wherein said recombinant Escherichia coli v erotoxin is a fusion protein comprising a non-veroto\ιn protein sequence and a poπion of the Eschei ichia coli verotoxin VT2 sequence 6 The method of Claim 1 wherein said subject is an adult 7 The method of Claim 1 wherein said subject is a child 8 The method of Claim 1 wherein said administering is parenteral 9 The method of Claim 1 wherein said administering is oral 10 A method of prophylactic treatment comprising a) providing: i) an antitoxin directed against at least one Escherichia coli verotoxin in an aqueous solution in therapeutic amount that is parenterally administrable, and ii) at least one subject is at risk of diarrheal disease: and b) parenterally administering said antitoxin to said subject 1 1 The method of Claim 10. wherein said subject is at risk of developing extra- intestinal complications of Escherichia coli infection. 12 The method of Claim 1 1. wherein said extra-intestinal complication is hemolvtic uremic syndrome. 13 A composition comprising neutralizing antitoxin directed against at least one Escherichia coli verotoxin in an aqueous solution in therapeutic amounts 14 The composition of Claim 13 wherein said Escherichia coli verotoxin is a recombinant toxin 15 The composition of Claim 14 wherein said recombinant Escherichia coli erotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion ol the Escherichia coli verotoxin VTl sequence. 16 The composition of Claim 14 wherein said recombinant Escherichia coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the Escherichia coli verotoxin VT2 sequence. 17 The composition of Claim 14 wherein said antitoxin is directed against a portion of at least one Escherichia coli verotoxin 18 The composition of Claim 14 wherein said portion of Escherichia coli is selected from the group consisting of subunit A and subunit B of VTl 19. The composition of Claim 14 wherein said portion of Escherichia coli is selected from the group consisting of subunit A and subunit B of VT2. 20. The composition of Claim 14 wherein said antitoxin is directed against a portion of at least one Escherichia coli verotoxin. 21. The composition of Claim 14 wherein said antitoxin is an avian antitoxin. 22. A method of treatment of enteric bacterial infections comprising: a) providing: i) an avian antitoxin directed against at least one verotoxin produced by Escherichia coli in an aqueous solution in therapeutic amount that is parenterally administrable. and ii) at least one infected subject: and b) parenterally administering said avian antitoxin to said subject. 23. The method of Claim 18 wherein said Escherichia coli is selected from the group consisting of Escherichia coli serotypes O157:H7. Ol:NM; O2:H5: 02:H7; 04:NM: O4:H10: O5:NM: O5:H16: O6:Hl; O18:NM: O18:H7; O25:NM: 026:NM: 026:H11: O26:H32: O38:H21: 039:H4: O45:H2: O50:H7: O55.H7: O55:H10: O82:H8: O84:H2: O91:NM: O91:H21: O103:H2: Olll:NM; Ol 11 :H8; Ol 11 :H30: 0111:H34: OH3:H7: O113:H21: O114.H48: O115:H10: O117:H4: O118:H12: O118:H30: O121:NM: O121.H19: O125:NM: O125:H8: O126:NM: O126:H8; O128:NM: O128:H2: 0128:H8: 0128:H12: O128:H25: O145:NM: 0125:H25: O146:H21: O153:H25: 0157.NM: O163:H19: 0165:NM: 0165:19: and O165:H25 24. The method of Claim 22 wherein said antitoxin comprises antitoxin directed against at least one Escherichia coli verotoxin. 25. The method of Claim 22 wherein said antitoxin is cross-reactive with at least one Escherichia coli verotoxin. 26. The method of Claim 22 wherein said antitoxin is reactive against toxins produced by members of the genus Shigella. 27. The method of Claim 26. wherein said antitoxin is reactiv e against toxins produced by Shigella dysenteriae. 28. A method for detecting Escherichia coli verotoxin in a sample comprising: a) providing: i) a sample: ii) an antitoxin raised against Escherichia coli verotoxin; and iii) a reporter reagent capable of binding said antitoxin: a d b) adding said antitoxin to said sample so that said antitoxin binds to the Escherichia coli verotoxin in said sample. 29. The method of Claim 28. wherein said antitoxin is an av ian antitoxin. 30. The method of Claim 28, further comprising the steps of: c) washing said unbound antitoxin from said sample: d) adding said reporter reagent to said sample so that said reporter reagent binds to said bound antitoxin: e) washing said unbound reporter reagent from said sample: and f) detecting said reporter reagent bound to said antitoxin bound to the Escherichia coli verotoxin so that the verotoxin is detected. 31. The method of Claim 30 wherein said detecting is selected from the group consisting of enzyme immunoassay, radioimmunoassav. fluorescence immunoassay. flocculation. particle agglutination, and in situ chromogenic assay. 32. The method of Claim 30 wherein said sample is a biological sample. 33. The method of Claim 30 wherein said sample is an environmental sample.

Inventors: CARROLL SEAN B, PADHYE NISHA V, STAFFORD DOUGLAS C, CARROLL, SEAN, B., PADHYE, NISHA, V., STAFFORD, DOUGLAS, C.
Applicants: OPHIDIAN PHARMACEUTICALS
Application year: 1996
Main classification: A61K39
IPC codes: A61K38, A61K39, A61P31, C07K14, C07K16, G01N33