The Experimental Study On Genetic Engineering Vaccine Of Multivalent Enterohaemorrhagic Escherichia Coli O157:H7

Since first being recognized as a human pathogen in 1982, enterohaemorrhagic Escherichia coli (EHEC) has gained increasing notoriety in the health and agricultural, it causes severe hemorrhagic colitis(HC), thrombotic thrombocytopenic purpura(TTP) and hemolytic uremic syndrome (HUS) of patients. Treatment of infection with E. coli O157 has been difficult because antibiotics do not change the course of the enteritis of E. coli O157 and may increase the incidence of HUS caused by the pathogen. This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis and release of intracellular Shiga toxins. So treatment that aim at this pathogen such as vaccines and antibody, are great significance to this disease . To evaluating vaccine, establishing a animal model infected with EHEC is very important. Porcine, cattle and sheep is sensitive to O157, but they are too big to adapt to researching. Mice is not sensitive to O157, we must enhance adherence to mice of O157 and reduce resistance to O157 of mice. E. coli O157:H7 has been shown to attach to the cytoplasmic membranes of intestinal epithelial cells, to efface their microvilli, and to cause actin to accumulate beneath sites of bacterial attachment. The eae gene, which has been shown to be necessary for attaching and effacing activity, encodes a 96-kDa outer membrane protein (OMP) which is termed Intimin. And Intimin is necessary for intimate attachment of the bacteria to epithelial cells . Intimin is reported to be highly immunogenic and there're studys showed that anti-Intimin antibodies can protect animals from colonization with EHEC O157:H7. The C-terminal fragment about 300 amino acid residue of Intimin (IntiminC300) is special and more suitable for fusion protein as vaccine. Stx1 and Stx2 are multimeric proteins displaying the classic AB5 structure seen in other bacterial exotoxins. Five Stx B subunits form a pentamer that recognizes globotriaosylceramide (Gb3-Cer) receptors found on many different eukaryotic cells. Upon host cell receptor ligation, the toxin is internalized, the A and B subunits dissociate, and the A subunit's N-glycosidase activity is activated, resulting in the removal of the adenine group from position 4324 in the eukaryotic28S rRNA of the 60S ribosomal subunit.The resulting A subunit-mediated inhibition of protein biosynthesisis cytotoxic to the target cell. Published evidence indicates a closer association between Stx2 expression by STEC and a more severe course of illness. So any potential STEC vaccine must generate a protective Stx2 immune response. Although animals immunized with Stx2 toxoid preparations are protected against a holotoxin challenge, there are safety concerns associated with using inactivated holotoxins in human vaccines, especially when the incidence of the disease is low. Therefore, if a practical Stx-based acellular STEC vaccine is to be developed, the atoxic Stx2B subunit would be a potential component. Almost all EHEC O157 have a large virulence plasmid, which encoded enterohemolysin (ehx, EHEC Hly). Hybridization studies revealed that EHEC Hly is related to E. coli α-hemolysin(α-Hly). Analysis of the DNA sequence revealed that 62.1% of EHEC hly nucleotide sequence was identical to the sequence of α-hly gene . Hly (cytolysins) were described to be important virulence factors of bacteria causing extraintestinal diseases and are active on different cells, such as lymphocytes, granulocytes, erythrocytes, and renal tubular cells. Hly is determined by the hlyCABD operon, hlyA gene encodes inactive prohemolysin, which is activated by HlyC, HlyB and HlyD are relative to secreting of HlyA. Five of the Mabs of α-Hly neutralized α-Hly hemolytic activity to varying degrees, three are located at (about amino acids 0 ～410). So it is worthful to researching immunoprophylactic potential of EHEC Hly. In view of these, this study investigated EHEC O157 vaccine from the following aspects: 1.Selecting EHEC O157 that have streptomycin-resistance by antibiotics selection (named O157-SMR), selecting EHEC O157-SMR that adapt to adhering and infecting Balb/c mice by separating it from stool of infected mice(named O157-SMR2). Balb/c mice , were given drinking water containing streptomycin to eliminate there normal microbiota for 3 days before and following oral challenge with O157-SMR2(1×1010CFU or 1×109CFU), and treated with clinical, microbiological and pathological examination. 33%～83% of mice were died 2～5d after infection, the O157:H7 can be detected in the feces of all alivemice over 13～22d. This Balb/c mice developed main clinical symptoms and pathological changes that people had after being infected by O157, the infection Balb/c mice with O157:H7 SMR2 is a useful model for studying pathogenicity and vaccine of EHEC. 2. Gene cloning, expression, primary purification and animal immunization of EHEC O157:H7 HlyAN436. The primers were designed and synthesized to amplify the HlyAN436 coding gene hlyaN436 by PCR. The target gene was cloned into pET-28a(+) plasmid through NcoI and XhoI restriction site after T-A cloning. Then the recombinant prokaryotic expression plasmid was transformed into E.coli BL21(DE3) and the target protein induced by IPTG was detected by PAGE. After primary purification, HlyAN436 was used to immunize two rabbits. As a result, HlyAN436 of EHEC O157:H7 was successfully expressed in prokaryotic expression system and a high titer antiserum against HlyAN436 has been gotten. 3. Gene cloning, expression, primary purification and animal immunization of EHEC O157:H7 Stx2B. The primers were designed and synthesized to amplify the Stx2B(deleted its leader peptides , amino acids 1～21) coding gene stx2b by PCR. The target gene was cloned into pET-11c plasmid through NdeI and BamHI restriction site after T-A cloning. Then the recombinant prokaryotic expression plasmid was transformed into E.coli BL21(DE3) and the target protein induced by IPTG was detected by PAGE. And prilimilary purified Stx2B by salting out and superdex filter chromatography. 4.Immunoprophylactic potential of the protein Stx2B, HlyAN436 and IntiminC300. In this study Balb/c mice were injected in the subscapular region with the preliminarily purified fusion protein the three antigens and Al(OH)3 adjuvant. The mice were injected 3 times, after the first vaccination, the two booster vaccinations were given every week thereafter. 7 days after each booster vaccination, few mice were phlebotomized from the tail and the special IgG titer of the sera were determinated by ELISA. 10 days after the second booster vaccination, 72 mice were infected with EHEC O157:H7 and the protective efficacy was observed thereafter. The result is that all these antigens have elicited high titer antiserum and has certain protective efficacy. Altogether, Balb/c mice model infected with streptomycin-resistance O157:H7 has been constructed achievely, this will be very useful to researching of genetic engineering vaccine of EHEC. Vaccine will perform the key role in controlling infection and epidemicoutbreak of EHEC O157:H7. It is important to study Stx2B, HlyAN436 and IntiminC300 protein in developing genetic engineered vaccine against EHEC O157:H7. There is no question that these vaccine against EHEC O157:H7 may benefit mankind all over the world in the future.