Identification Of The Monoclonal Antibodies Against The Main Protective Antigens Of EHEC O157:H7

Enterohemorrhagic Escherichia coli(EHEC) O157:H7 is a new pathogen first detected in America at 1982, which is responsible for dehydrating infantile diarrhea, hemorrhagic colitic and the hemolytic uremic syndrame. There had been several outbreaks of EHEC O157 infection in Japan, America, England, Canada, Australia and so on in past ten years. The severest outbreak was in Japan during May to August in 1996, above 9000 humans were infected. By epidemic investigation, animals especially the ruminant such as cattle and sheep are natural reservoir of EHEC O157, the contaminated water, vegetables and meats are main infection sources. In China, O157 strains had been isolated from humans and many kinds of animals in Jilin, Fujian, Gansu, Zhejiang, Anhui province, etc. Because of high infectivity and acid tolerance, only 100-200 O157 bacteria can breakthrough gastric acid barricade, breeding in intestinal tract in a large number, inducing human infection, but the other pathogenic Escherichia coli request 1×106 bacteria. EHEC O157 gets more and more high attention all over the world due to its high virulence, severe residual, bad prognosis and without effective treat means. For antibiotic treatment to O157 can lead to severe disturbance of normal microbial population in intestinal tract and create drug resistance strains. And some antibiotic can schizolysis thallus, cause toxin releasing and increase danger to organism. The majority of specialists did not claim applying flushing dose orally antibiotic to treat O157 infection. Thus, active precaution make more important role in O157 infection. On the one hand, the effective vaccines should be developed to degrade human's susceptivity and abscise conveying from animal to human. On the other hand, the rapid detection methods should be estabished to detect O157 from the contaminated foods and water sources. Monoclonal antibodies targeted to bacterial virulence factors and protective antigens provide important application value for bacterial vaccine and rapid detection. EHEC O157 has two important virulence factors, the one is the intestine adherence factor(Intimin) encoded by eae gene in the pathogenicity island (PAI) termed the locus of enterocyte effacement (LEE), the other is the Shiga-toxin(Stx) encoded by lysogenic lambdoid bacteriophages. Both of Intimin and Stx are effective protective antigens, which have been researched for EHEC precaution and detection all over the world. Because Intimin and Stx are hard to express and to be purified in EHEC O157 wide type strains, monoclonal antibodies against Intimin and Stx were seldom developed now. We used the purified fusion protein to immunize mice, and got three monoclonal antibodies which were specifically targeted to Intimin and Stx1, Stx2 respectively. The monoclonal antibodies can be used to determine and evaluate the virulence factors expression state in EHEC O157 deletion mutation strains, or to detect and identify EHEC O157 wide type strains. With constructed recombination expression strains BL21(pET28::stx1/2B) and BL21(pET28::eae-stx1/2B), we prepared the fusion proteins expressed in inclusion bodies. The fusion proteins Stx1/2B and Eae-Stx1/2B were purified by SDS-PAGE separation and electroelution. The concentrations of purified proteins were 0.13mg/mL and 0.25mg/mL respectively. BALB/c mice were intraperitoneally immunized with purified protein Eae-Stx1/2B, and the splencytes of immunized mice were fused with myeloma cells Sp2/0. Hybridoma cells were screened by indirect ELISA and monoclonal hybridoma cells were obtained using limited dilution. Three hybridomas, named 1G2, 3C6 and 1B10, were successfully established. By ELISA, the titers of hybridoma culture supernatants were 1:6400, 1:8000 and 1:160 respectively, and the titers of purified ascitic fluids were 1:6.4×106, 1:1.2×107 and 1:3.2×104, protein concentrations of purified ascitic fluids were 5.66mg/mL, 6.46mg/mL and 3.27mg/mL respectively. Identification of subclass showed that 1G2 belonged to IgG1, 3C6 belonged to IgG2b and 1B10 belonged to IgM respectively. By western-blot analysis, three monoclonal antibodies only reacted with fusion proteins specifically. 1G2 only reacted with Eae-Stx1/2B, 3C6 and 1B10 reacted with Eae-Stx1/2B and Stx1/2B, what confirmed that 1G2 was targeted to Intimin, 3C6 and 1B10 were targeted to Stx. Three hybridomas were stable to secret antibody through three-month continuous culture and six-month -80℃cold-storage.