Research Of The Recombinant Attenuated Escherichia Coli Oral Vaccine Candidate Strain Based On The Insertion Of Chimeric Enterotoxin Gene In The Pseudogene

Enterotoxigenic Escherichia coli（Enterotoxigenic Escherichia coli,ETEC）is a severe newborn piglets infectious disease.Piglets commonly suffer from severe diarrhea dehydration and leading to death,caused huge economic losses.ETEC induced diarrhea mainly through adhesinand 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、yaiX、yge and yge Q、WbbL、insN、eaeH+ and ykgA,PCR amplify identified that E.coli 0142:△STa only has yaiT and yaiX pseudogenes position for foreign genes insertion.To identify the insertion position of mosaic gene to the natural adjuvant LT1 structure,a LT1 specific neutralizing activity monoclonal antibody was prepared.The antibody is effectively blocking the adhesion of LT1 toxin to GM1 receptor.GM1 recptor combination peptide MBP-LT1B-7（61QKKAIERMKDTLRITY76）was screened through bioinformatics software ABCpred prediction and LT1B sectional monoclonal antibody screen.The pseudogene yaiT and yaiX homologous arms was amplified with its own template,cloned into pDOC-K plasmid to construct the PRPL-Kil double screen plasmid,then the PRPL-Kil and pACBSCE plasmid was transformed to attenuated E.coli 0142:△STa.After the inducing of L-arabinose,pACBSCE 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 yaiT and yaiX,which could homologous recombinant with pseudogene yaiT and yaiX gene of E.coli 0142:△STa to gain the double selection platform E.coli 0142（yaiT::PRPL-Kil）and 0142（yaiX::PRPL-Kil）.PRPL-Kil operon of E.coli O142（yaiT::PRPL-Kil）and 0142（yaiX::PRPL-Kil）did not express at low temperature,but express at 43℃,and the expressed product could kill the E.coli strain for counter selection.A subunit of LT1 operon was mutated from LT1R192G by SOE-PCR method for generating the LT192-STa₁₃.STb genes were inserted into end of B subunit of LT1₁₉₂-STai3 operon for generating the LT1₁₉₂-STa₁₃-STb.LT1A was divided into LT1A1 and LT1A2,cloning STa₁₃-STb from LT1₁₉₂-STa₁₃-STb,then inserted into end of LT1A1 generating the LT1A1-(STa₁₃-STb).Subsequently,assembly LT1A1-(STa₁₃-STb)and LT1A2-LT1B-STa₁₃-STb generating the LT1A1-(STa₁₃-STb)-LT1A2-LT1B-(STa₁₃-STb)operon,respectively.PRPL-Kil operon of E.coli 0142（yaiT::PRPL-Kil）and E.coli 0142（yaiX::PRPL-Kil）platform was replaced by LT1A1-(STa₁₃-STb)-LT1A2-LT1B-（STai3-STb）operon for constructing the recombinant strains E.coli ER-T and E.coli ER-X.Analyzed by toxicity test,survival properties in imitative gastrointestinal environments,safety test,bacterial growth test,heredity stability test,fimbrial growth test and colonization ability in intestinal tracts.Showed that E.coli ER-T and E.coli 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 IgG level in the serum,and the IgA level in fecal material and intestine mucus of the immunized mice were determined and detected by ELISA.The results showed that anti-LTIA（P<0.05）,anti-LT1B（P<0.05）,anti-STa（P<0.05）,anti-STb（P<0.05）and anti-F41（P<0.05）IgG antibodies were detected in the serum of immunized mice on the days 7 to 21.Anti-LT1A（P<0.05）,anti-LTIB（P<0.05）,anti-STa（P<0.05）,anti-STb（P<0.05）and anti-F41（P<0.05）IgA antibodies were detected in the fecal of immunized mice on the days 7 to 28.Anti-LTIA（P<0.05）,anti-LT1B（P<0.05）,anti-STa（P<0.05）,anti-STb（P<0.05）and anti-F41（P<0.05）IgG and IgA 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-y and IL-4 ELISA showed a marked shift towards Th2-mediated immunity in mesenteric lymph node cells.Indicating that oral immunization stimulate system Thl immune response was lower than stimulated local mucosal immune response in mice.Results of in vitro 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 E.coli ER-T and E.coli ER-X had a good protective effect to the mice.After experimental vaccination to the piglets and pregnant pigs with recombinated strains E.coli ER-T and E.coli ER-X,the IgG and IgA antibody titer,cell proliferation,cytokine,in vivo and in vitro neutralizing assay were carried.The data shown that the protective 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,LU1 and GM1 receptor binding site was identified through epitope screen.Recombinated E.coli strains E.coli ER-A and E.coli 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.