| Enterotoxigenic Escherichia coli(Enterotoxigenic Escherichia coli, ETEC) is a severe newborn piglets infectious disease. Piglets commonl y suffer from severe diarrhea dehydration and leading to death, caused huge economic losses. ETEC induced diarrhea mainly through adhesin and enterotoxin. According to the latest epidemiological E. coli studies, with the resistance rate is improving, adhesions detection rate decline. Therefore, traditional medicines and vaccines could not provide therapeutic or vaccination effect against pathogen adhesion, demanding a new effective ETEC vaccine which could prevent piglet diarrhea.According to the sequence of E. coli strain K12, Applied Biology software DNAstar to screened predictation pseudogene location may carrying foreign gene include yaiT、yai X、yge and yge Q、wbb L、insN、eae H+ and ykg A. PCR amplify identified that E. coli O142:△STa only has yai T and yaiX pseudogenes position for foreign genes insertion.To identify the insertion position of mosaic gene to the natural adjuvant LT-1 structure, a LT-1 specific neutralizing activity monoclonal antibody was prepared. The antibody is effectively blocking the adhesion of LT toxin to GM1 receptor. GM1 recptor combination peptide MBP-LTB-7(61QKKAIERMKDTLRITY76) was screened through bioinformatics software ABCpred prediction and LTB sectional monoclonal antibody screen.The pseudogene yaiT and yaiX homologous arms was amplified with its own template, cloned into p DOC-K plasmid to construct the PRPL-Kil double screen plasmid, then the PRPL-Kil and p ACBSCE plasmid was transformed to attenuated E. coli O142: △STa. After the inducing of L-arabinose, p ACBSCE plasmid could express the I-Sce I enzyme and the Red recombinose. I-Sce I enzyme cleaved the PRPL-Kil double screen plasmid vector for generating the PRPL-Kil double screen gene flanked and homologous arms of yai T and yaiX, which could homologous recombinant with pseudogene yai T and yai X gene of E. coli O142: △STa to gain the double selection platform O142(yai T::PRPL-Kil)and O142(yai X::PRPL-Kil). PRPL-Kil operon of O142(yai T::PRPL-Kil)and O142(yai X::PRPL-Kil)did not express at low temperature, but express at 43°C, and the expressed product could kill the E. coli strain for counter selection.A subunit of LT operon was mutated from LTR192 G by SOE-PCR method for generating the LT192-STa13. STb genes were inserted into end of B subunit of LT192-STa13 operon for generating the LT192-STa13-STb. LTA was divided into LTA1 and LTA2, cloning STa13-STb from LT192-STa13-STb, then inserted into end of LTA1 generating the LTA1-(STa13-STb). Subsequently, assembly LTA1-( STa13-STb) and LTA2-LTB-STa13-STb generating the LTA1-( STa-STb)-LTA2-LTB-(STa-STb)operon, respectively. PRPL-Kil operon of O142(yaiT::PRPL-Kil)and O142(yai X::PRPL-Kil)platform was replaced by LTA1-(STa-STb)-LTA2-LTB-(STa-STb)operon for constructing the recombinant strains ER-T and ER-X.Analyzed by toxicity test, survival properties in imitative gastrointestinal environments, safety test, bacterial growth test, heredity stability test, fimbrial growth test and coloni zation ability in intestinal tracts. Showed that ER-T and ER-X were safe, stability, colonize the intestinal tract of immunized mice and piglets.BALB/c mice were immunized via oral with ER-T and ER-X. The Ig G level in the serum, and the Ig A level in fecal material and intestine mucus of the immunized mice were determined and detected by ELISA. The results showed that anti-LTA(P < 0.05), anti-LTB(P < 0.05), anti-STa(P < 0.05), anti-STb(P < 0.05)and anti-F41(P < 0.05)Ig G antibodies were detected in the serum of immunized mice on the days 7 to 21. Anti-LTA(P < 0.05), anti-LTB(P < 0.05), anti-STa(P < 0.05), anti-STb(P < 0.05)and anti-F41(P < 0.05)Ig A antibodies were detected in the fecal of immunized mice on the days 7 to 28. Anti-LTA(P < 0.05), anti-LTB(P < 0.05), anti-STa(P < 0.05), anti-STb(P < 0.05) and anti-F41(P < 0.05)Ig G and Ig A antibodies were almost detected in the lac feminium, spleens, intestinal mucus and mesenteric lymph nodes samples on day 42. The results of spleen lymphocyte proliferation and mesenteric lymphocyte proliferation showed that recombinant proteins and enterotoxins stimulated the potent production of specific T-lymphocytes. The results of IFN-γ and IL-4 ELISA showed a marked shift towards Th2-mediated immunity in mesenteric lymph node cells. Indicating that oral immunization stimulate system Th1 immune response was lower than stimulated local mucosal immune response in mice. Results of in vit ro and in vivo tests showed that serum, spleens, intestinal mucus and mesenteric lymph nodes samples collected from immunized mice demonstrated obvious inhibition to enterotoxins. These results showed that ER-T and ER-X had a good protective effect to the mice.After experimental vaccination to the piglets and pregnant pigs with recombinated strains ER-T and ER-X, the Ig G and Ig A antibody titer, cell proliferation, cytokine, in vivo and in vitro neutralizing assay were carried. The data shown that the prote ctive effect was similar to mice experiment. In the vaccination and challenge experiment, the intestinal villus of vaccinated group has shown significant various advantages compared to control group(P < 0.05), suggesting vaccination protected the piglet intestinal tract to the direct attack of enterotoxins.In order to enhance the vaccination effect of recombinated live attenuated E. coli vaccine, LT1 and GM1 receptor binding site was identified through epitope screen. Recombinated E. coli strains ER-A and ER-B which could stable express chimeric enterotoxin was constructed. The difference between pseudogene carrying exogenous was explored. This study provides the experimental data for the development of recombination attenuated E. coli oral vaccine. |
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