| Immunological tolerance induced by oral administration antigen can suppress and delay the auto-reactive immune disease and hypersensitivity. However, this approach requires administration of massive of antigens, which are then only effective in rather narrow dose ranges. Oral tolerance is especially difficult to induce in an already immunological sensitized host. The nontoxic B subunit of the cholera toxin (CTB) was used to overcome such limitations by serving as a carrier molecule for chemically conjugated autoantigens for the induction of oral tolerance. Application of CTB as a carrier has been demonstrated for prevention and treatment of autoimmune diabetes in NOD(non-obese diabetic) mice, and the dose was 500-5000 times lower than these treat with unconjugated insulin. The genetically engineered fusion proteins have clear advantages over chemicals conjugates. The composition of the genetically engineered fusion protein is invariable. and the processing a single protein is much easier than producing multiple biological components that subsequently be chemically linked.The gene of proinsulin and the GDA (531-545) peptide were fused to the 3' end of the CTB by overlapping PCR to obtain the fusion genes CTB-lnsB(CI) and CTB-GAD(CG). Then the fusion genes were subcloned into the pET22b vector to construct the recombinant plasmids, CI-pET22b and CG-pET22b. The two plasmids were transformed into BL21(DE3) and induced by lactose. The fusion genes were expressed and accumulated as inclusion bodies. The molecular weight of CI and CG were 14kD and 17kD, respectively. The CI accumulated to the level of 35% total bacterial proteins and the CG accumulated to 30%. The inclusion bodies washed three times by the 2M urea solution and denatured by 8M urea solution. The denatured proteins were refolded in vitro by dilution, then the proteins were purified. The Western-blotting results indicated that both CI and CG... |
PDF Full Text Request