Oral Attenuated Salmonella Enterica Serovar Typhimurium Vaccine Expressing Multivalent Enterohae-morrhagic Escherichia Coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) has gained increasing notoriety in the health and agricultural, since first being recognized as a human pathogen in 1982. 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 .The interplay between bacterium and ruminant host probably has its main role in promoting infection and pathopoiesis. The adherence of EHEC on intestinal epithelial cells is believed to be the first step for developing these diseases. The adhesion is mainly mediated through the type III secretion system. The translocon apparatus is initially an extended structure primarily made up of EspA. This protruding organelle may be necessary to bypass microvilli or mucus layers. EspA makes filamentous appendages surrounding the bacterium and forms a needle structure through which various proteins including Esp (E. coli secreted protein) proteins and Tir are secreted. These secreted proteins drive the intimate association of the bacterium with the host epithelial cell, resulting in histological changes of epithelial cells, which is called attaching-and-effacing (A/E) lesion including F-actin aggregation. EspA not only has initial role in adherence of O157 with host cell, but also build a bridge of effect protein translocation between them. Consequently, study on biological action of EspA is very important in controlling, preventing and curing the infection of O157.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.Recent progress in the knowledge of the genetics of Salmonella virulence and modern recombinant DNA technology offers the possibility to introduce multiple defined attenuating and irreversible mutations into the bacterial genome. Live attenuated Salmonella vaccines were used for the expression of heterologous antigens/proteins that can be successfully delivered to the immune system. Furthermore, Recombinant attenuated Salmonella strains are also an attractive means of delivering heterologous antigens to the immune system, thereby, stimulating strong mucosal and systemic immune responses and consequently provide an efficient platform technology to design novel vaccination strategies.In this study ,we use the asd+ balanced lethal system with no antibiotic selection to express the specific protein.In view of these, to develop an oral attenuated Salmonella typhimurium vaccine against the infection of O157 and to evaluate its efficacy in mice. The study has been involved in the following aspects:1. PCR was performed to get a single gene EspA and fusion gene EspA-IntiminC300 and EspA-IntiminC300-Stx2B. These three gene were then subcloned into the plasmid pYA3149, and the recombinant plasmid were then transformed into attenuated Salmonella typhimurium x4550 respectively.2. The recombinant plasmid were induced with IPTG at 37℃.They were confirmed by SDS-PAGE and western blotting with specific antibodies.The target protein were expressed.3. The stability of the plasmid in the bacteria and the immune responses was analyzed.4. Balb/c mice were orally immunized with the recombinant Salmonella typhimurium x4550. The mice were immunized every 2wk. To reinforce, after three times orally immunization, the mice were immunized subcutaneous one time. After three times orally immunization, serum titer of antibody was detected by ELISA, and after subcutaneous 10 days, serum titer of antibody was detected by ELISA too. The level of serologic specific IgG assessed by ELISA indicated that immunized groups have a better level compared with PBS control group (p<0.001),and the boost groups have a better level compared with the oral groups. After the last immunization, mouse were challenged with 109CFU O157:H7 strain after 15 days,and the result showed the protective efficacies of oral the groups were35%,44% and 62% respectively, the boost groups were increased to 56%,67%,89% respectively.To sum up, it is important to investigate an effect vaccine to control the spread of O157. The attenuated salmonella as an effective vaccine for the infection of O157 is very promising.