OBJECTIVE To develop a trivalent genetically engineered inactivated Escherichia coli vaccine (K88ac-3STa-LTB) that neutralizes the STa toxin by targeting fimbriae and entertoxins for the treatment of enterotoxigenic E coli. ANIMALS 18- to 22-g mice, rabbits, pregnant sows. PROCEDURES Using PCR, the K88ac gene and LTB gene were cloned separately from the template C83902 plasmid. At the same time, the 3 STa mutant genes were also amplified by using the gene-directed mutation technology. Immune protection experiments were performed, and the minimum immune dose was determined in mice and pregnant sows. RESULTS The ELISA test could be recognized by the STa, LTB, and K88ac antibodies. Intragastric administration in the suckling mouse confirmed that the protein had lost the toxicity of the natural STa enterotoxin. The results of the immune experiments showed that K88ac-3STa-LTB protein could stimulate rabbits to produce serum antibodies and neutralize the toxicity of natural STa enterotoxin. The efficacy test of the K88ac-3STa-LTB-inactivated vaccine showed that the immune protection rate of the newborn piglets could reach 85% on the first day after suckling. At the same time, it was determined that the minimum immunization doses for mice and pregnant sows were 0.2 and 2.5 mL, respectively. CLINICAL RELEVANCE This research indicates that the K88ac-3STa-LTB trivalent genetically engineered inactivated vaccine provides a broad immune spectrum for E coli diarrhea in newborn piglets and prepares a new genetically engineered vaccine candidate strain for prevention of E coli diarrhea in piglets.

In order to develop the K88ac-3STa-LT2 trivalent genetically engineered inactivated vaccine, the recombinant strain BL21(DE3)(pXK88ac3STaLT2) was constructed. ELISA test showed that the K88ac-3STa-LT2 fusion protein could be recognized by STa monoclonal antibody, LT2 and K88ac antibody. The test of intragastric administration in the suckling mouse confirmed that the expressed fusion protein had lost the toxicity of the natural STa enterotoxin. The results of immune experiments showed that K88ac-3STa-LT2 fusion protein could induce rabbits to produce serum antibodies, which had the effect of neutralizing the toxicity of natural STa enterotoxin. Efficacy test of the K88ac-3STa-LT2 genetically engineered inactivated vaccine showed that the immune protection rate of the newborn piglets could reach 85% on the 1st day after suckling. At the same time, it was determined that the minimum immunization doses for mice and pregnant sows were 0.2 mL and 2.5mL respectively. This study provided scientific parameters for the industrialized production of the vaccine and showed that the K88ac-3STa-LT2 trivalent genetically engineered inactivated vaccine had a broad immune spectrum for E. coli diarrhea in newborn piglets. Through this research, a new genetic engineering vaccine candidate strain was provided for more effective prevention of E. coli diarrhea in piglets.

In order to develop the K88ac-3STa-LT2 trivalent genetically engineered inactivated vaccine, the recombinant strain BL21(DE3)(pXK88ac3STaLT2) was constructed. ELISA test showed that the K88ac-3STa-LT2 fusion protein could be recognized by STa monoclonal antibody, LT2 and K88ac antibody. The test of intragastric administration in the suckling mouse confirmed that the expressed fusion protein had lost the toxicity of the natural STa enterotoxin. The results of immune experiments showed that K88ac-3STa-LT2 fusion protein could induce rabbits to produce serum antibodies, which had the effect of neutralizing the toxicity of natural STa enterotoxin. Efficacy test of the K88ac-3STa-LT2 genetically engineered inactivated vaccine showed that the immune protection rate of the newborn piglets could reach 85% on the 1st day after suckling. At the same time, it was determined that the minimum immunization doses for mice and pregnant sows were 0.2 mL and 2.5mL respectively. This study provided scientific parameters for the industrialized production of the vaccine and showed that the K88ac-3STa-LT2 trivalent genetically engineered inactivated vaccine had a broad immune spectrum for E. coli diarrhea in newborn piglets. Through this research, a new genetic engineering vaccine candidate strain was provided for more effective prevention of E. coli diarrhea in piglets.