Prokaryotic Expression Of Shiga Toxin2e Gene Mutant And Study Of Immunobiological Properties Of Recombinant Proteins

Edema disease (ED) of swine is an acute deadly disease of weaning piglets, it results from E.coli producing Shiga toxin2e (Stx2e). Stx2e is the major toxic factor of E.coli which can result in Edema disease of swine. Antitoxic immunity can protect animals from infection of toxigenic E. coli, and it already became one of the focal point of ED study. The extraction of toxin or toxoid is the key of the mechanism and effect of antitoxic immunity study. There are two methods of toxoid preparation:One method is inactivate natural Stx2e by chemical method, but the toxoid prepared by this method can produce side-effects of disturbing weight increase or leading to weight decrease of animals; The other method is mutate the enzyme activity sites of Stx2e to obtain toxoid by genetic engineering, and this kind of toxoid doesn’t need to inactivate by chemical method or effect animals’weight or other production properties.According to the A、B gene of Stx2e from GenBank, four pairs of primers were designed. The one of the four pairs was used to amplify the sequence of B subunit deleting its signal peptide. Two pairs of primer was used to amplify genetically mutative Stx2e A subunit by PCR, introducing double point mutations at residues167(Glu-Gln) and170(Arg-Lys). The final pair of primer was used to amplify the sequence of mutative Stx2eA subunit deleting its signal peptide using overlap extension PCR technique. The PCR products were cloned into cloning vector pMD18-T simple vector, and resulting recombinants were called pMD18-T-Amu and pMD18-T-B. The sequencing result showed that the nucleotide homology of A unit was99.0%～99.6%and amino acid homology was98.3%-99.0%compared with the Stx2eA gene from GenBank, and succefully got point mutations at residues167(Glu-Gln) and170(Arg-Lys); The nucleotide homology of B unit was99.5%～100%and amino acid homology was98.6%-100%compared with the Stx2eB gene from GenBank.Collect the recombinant plasmids pMD18-T-Amu and pMD18-T-B, and obtain Stx2eAmu and Stx2eB gene sequences by enzyme digestions. And then they were recombinated into prokaryotic expression vector pET Duet-1(named pET-ABmu). The recombinant plasmids were transformed into E.coli BL21(DE3) and induced by IPTG. The result of SDS-PAGE analysis showed that Stx2eAmu and Stx2eB gene was successfully expressed in E.coli BL21(DE3). The molecular weights were35kDa and7.5kDa (named His-Stx2eAmu and Stx2eB) and the recombination proteins were expressed with the form of inclusion body. The immunity reactivity of the recombinant proteins was confirmed by Western-blot.The purified proteins His-Stx2eAmu and Stx2eB were used for immunogen to inject into ICR mice celiac cavity, in order to test the toxicity and immunogenicity of recombinant proteins, and then challenge with minimum lethal dose Stx2e crude extract to observe immunity protection of recombinant proteins to mice; Test the cytotoxicity of recombinant proteins to Vero cells and neutralization titer of immune serum against Stx2e. The result showed that ICR mice immunized with recombinant proteins don’t have any symptoms, which demonstrate the recombinant proteins were safe with ICR mice, and intraperitoneal injection can induce organism to produce effective protective antibody; Purified recombinant His-Stx2eAmu and Stx2eB were diluted to inoculate Vero cells, and there was no appearance cytopathic effect by His-Stx2eAmu and Stx2eB, but it can extend the time of cell monolayer formation.