Study On Biological Properties And Immunoprotection Of Enzyme Active Sites Mutated Shiga Toxin2e

Edema disease (ED) of swine is an acute deadly infectious disease of weaning piglets, it results from certain serotype E.coli which colonizes in small intestine and producing Shiga toxin2e (Stx2e). Edema disease (ED) of swine is also called enterotoxemia. The major characteristic of the disease is greater curvature, mesentery and brain edema, and usually is associated with neurological sign, for example, ataxia, convulsions or paralysis. The pathogenic factor of ED is complex, its primary virulence factor is F18fimbriae and Stx2e.pET-ABmu E.coli is a kind of recombinant bacteria which was structured by my laboratory to express Stx2eAmu and Stx2eB proteins at the same time. Under the condition of37℃and0.5mmol/L IPTG, the recombinant bacteria expressed maximum Stx2eAmu and Stx2eB proteins. The recombinant proteins mainly in the form of inclusion body, and the molecular weights were35kDa and7.5kDa. 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 weaning piglets by intramuscular injection at neck. The immune piglets can produce antibody after7days, and the amout of the antibodies can reached highest after42days. The serum antibody titer was1:5120. We performed the neutralization test by immune serum and the crude Stx2e protain in vero cells, and the neutralization titer of immune serum against Stx2e is26. Tt was proved that the serum of immune piglets can restrain Stx2e observably.The purified proteins His-Stx2eAmu and Stx2eB were used for immunogen to inject into three batches ICR mice celiac cavity, to text 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. The result showed that ICR mice immunized with recombinant proteins don’t have any symptoms, which demonstrate the recombinant proteins were safe to ICR mice. The challenge assay by minimum lethal dose Stx2e crude extract to ICR mice proved that ICR mice can produce protective antibodies effectively by intraperitoneal injection. The average immune protective rate can reach93%and more, and the average immune protective rate to two minimum lethal doses can reach90%. The recombinant proteins His-Stx2eAmu and Stx2eB can generate well immunity protection.The purified proteins His-Stx2eAmu and Stx2eB were used for immunogen to inject into three batches weaning piglets of35days old by intramuscular injection at neck, to test the immunity protection of the recombinant proteins to pigs, and then challenge with minimum lethal dose Stx2e crude extract to observe immunity protection of recombinant proteins. The result showed that weaning piglets immunized with recombinant proteins don’t have any symptoms, which demonstrate the recombinant proteins were safe to weaning piglets; In the challenge assay by minimum lethal dose Stx2e crude extract to weaning piglets, one pig had symptoms in experimental group, and other piglets had no symptom, so the incidence rate is8.3%; In the control group, all the4piglets had symptoms, the incidence rate is100%, and3piglets died, the death rate is75%. It proved the weaning piglets can produce protective antibodies effectively by intramuscular injection at neck. The recombinant proteins His-Stx2eAmu and Stx2eB can generate well immunity protection.