The Study Of Enterobacterial O-specific Polysaccharides Bio-conjugate Vaccines

Dysentery and cholera, which are caused by Shigella spp. and Vibrio cholerae, are both important intestinal infectious diseases. The conventional treatment for dysentery and cholera depends on the use of antibiotics. But the abuse of antibiotics also enhances the bacterial resistance and brings multidrug-resistant strains. Currently, vaccination is the most economical and effective means of prevention. But no ideal, safe and effective vaccine against Shigella spp. has been developed now. The recombinant B subunit/cholera vaccine against Vibrio cholerae O1 group is used for prevention. There is no prevent vaccine against O139 group. Thence, it is great important to research the polysaccharide conjugate vaccine.With using the polysaccharide’s immunogenicity, people get the polysaccharide vaccine, which is the T-cell independent antigen. The T cell don’t participate in polysaccharide vaccine’s immune process. The body produce lower affinity antibody with no immune memory. Binding the bacterial polysaccharide with a suitable carrier, people get the glycoprotein vaccine, which is the T-cell dependent antigen. The glycoprotein vaccine can cause the recognition of both T cell and B cell. So it improves the immune effect significantly, with body’s immune memory. It is similar between the synthesis of glycosylated protein and bacterial lipopolysaccharide. Genes associated with protein glycosylation has been found in bacteria recently, such as pgl gene cluster. All these make the binding of O polysaccharide and protein carrier possible in vivo. The glycoprotein vaccine made by biological synthesis needs little money and simple operation. By only one purification, people can get the target glycoprotein. Also it has good uniformity because people know the glycosylation sites clearly.According to the previous studies in our laboratory, we had successfully constructed waa I deletion mutant of Shigella flexneri 2a 301. The gene waa I is the O-antigen ligase in the biosynthesis of lipopolysaccharide(LPS). Moreover, we constructed a genetically modified Shigella strain expressing O-specific polysaccharides(OPS) conjugated to a carrier, which was cholera toxin B subunit(CTB). Then, the expression was induced, and been tested by SDS-PAGE and western blot. The results showed that, the CTB and OPS was combined in vivo. Next, based on the characteristic of the substrate and His-tagged protein, the conjugate(CTB-OPS) was further purified through affinity chromatography and ion exchange chromatography. And we detected that it was in the form of sugar polymer state. Then, the conjugate was evaluated for their immunogenicity. We chose the mice as test animals which were injected an amount of 2.5 μg purified glycoproteins, and with the control of OPS group and adjuvant group. The results of ELISA showed that the conjugate evoked higher titers of Ig G than by O-specific polysaccharides, suggesting that CTB increased immunogenicity of OPS significantly as a carrier. The conjugate stimulated the mice generating Ig G1, Ig G2 a, Ig G2 b and Ig G3 antibodies. Among these Ig G1 antibody was the most. The conjugate could cause the body generating both cellular immune response and humoral immune response. And the humoral immune response was principal. Finally, the wild strains were injected to the mice of each group, with the minimum lethal dose of poison. After a week, the mice of the OPS group, the adjuvant group and the control group all died. There were mice survived in the glycoprotein groups. So it illustrated that Ig G antibody was the protective antibody. As a vaccine, the CTB-OPS glycoprotein could protect the body from the invasion of Shigella to a certain degree. This study successfully prepared Shigella O polysaccharide protein conjugate vaccine as a candidate vaccine using biological synthesis. In addition, the study also found that the wild-type strain could also express the glycoprotein with the glycosylation system. The sugar types of the two glycoprotein(produced by wild-type strain and waa I deletion mutant) were slightly different. But the serum titer after immunization in mice caused no significant difference between the two groups. This provides a new way for our study. When the knockout strain is not so easy to construct, we can try to import glycosylation system into the wild-type strain, and detect if the glycoprotein is expressed or not.In this study, a suicide vector p WM91 was used to construct cholera O139 group 93-3 waa L deletion strain. Then, the study showed that the physiological and biochemical characteristics were the same in the 93-3 and 93-3△waa L. Then we can import the glycosylation system to the deletion strain. This provides a basis for glycoprotein vaccine against Vibrio Cholerae O139 group.