Construction Of Targeting Lactobacillus Casei Expression F4~+ETEC And Immunogenicity Analysis

Enterotoxigenic Escherichia coli is a major pathogens of piglets diarrhea and also animportant incentive for it, Which F4~+ETEC occupys obvious dominance. Therefore control F4~+ETEC infection becomes an important measure to prevent piglets diarrhea. Piglets diarrhea is along-term problem that troubled farms and farmers, which not only brings direct death losses andhuge amounts of medication cost, but also indirectly affect the growth rate for the next phase of therehabilitation pig rearing rate. It’s very serious harm to became important constraints which affectthe economic benefits of the pig industry worldwide.In this study, We first cloned F4~+ETEC FaeG, STa-LTB, STb gene and then obtain theoptimal combination of new F4~+ETEC protective antigen gene of STa-LTB-STb-FaeG and M celltargeting gene of STa-LTB-STb-FaeG-co1by SOE-PCR, after that construction recombinant E.coli which express FaeG, FaeG-co1, Sta-LTB-STb-FaeG or STa-LTB-STb-FaeG-co1. SDS-PAGEand Western blot analysis showed that the fusion gene was highly expressed in E. coli and mainlyin the form of inclusion body. Mice were immunized with the inclusion body of the recombinantproteins, respectively, then detect the level of specific IgG and sIgA antibody by ELISA and thenchallenged with the ETEC cvcc230. The results showed that the target protein without toxicity caninduce a protective immune response, The specific IgG antibody level of M cell target peptidemodified group were significantly higher than the unmodified group and M cell target peptidemodified group showed100%survival rate when challenged to ETEC. These results suggest thatthe protective antigen is safe and effective and the M-cell targeting peptide has immuneenhancement function to the antigen. Use the immune protective antigen protein FaeG, FaeG-co1,STa-LTB-STb-FaeG anf STa-LTB-STb-FaeG-co1gene as target gene, then cloned into aLactobacillus expression vector pPG-2, eletroporated into L.casei. Construction recombinantLactobacillus casei expression system that expression F4~+ETEC the immune protective antigenprotein. The recombinant bacteria named pPG-2-FaeG/L.casei393, pPG-2–FaeG-co1/L.casei393,pPG-2-Sta-LTB-STb-FaeG/L.casei393, pPG-2-STa-LTB-STb-FaeG-co1/L.casei393. Therecombinant L.casei are induced by1%lactose, western blot showed that the gene was expressed inL.casei.We do immunogenicity preliminary assessment using BALB/c mice, for investigating potential using value of the recombinant L.casei system of F4~+ETEC the immune protectiveantigen protein. Orally inoculated mice with10~9viable cells amount, collected fresh faeces, blood,nasal and external genital lavage fluid samples in different times. Then we were measured specificsIgA and serum IgG by ELISA. And detected the expression level of Th1, Th2, Th17by flowcytometry method, the spleen lymphocyte proliferation by MTT.Oral immunization results show that one dose immunization group pPG-2-FaeG/L.casei393and pPG-2-FaeG-co1/L.casei393could detect remarkable special IgG in the serum of mice, specialsIgA level in the stool, vaginal lavage of mice. Compared with the control group, discrepancysignificant predominance(P<0.01), M cell-targeting peptide modified group pPG-2-FaeG-co1/L.casei393significantly higher than the unmodified group. Three immunization group pPG-2-SLSF/L.casei393and pPG-2-SLSF-co1/L.casei393could detect remarkable special sIgA level inthe stool, vaginal and nasal lavage of mice, significant differences between the groups(P<0.01).Compared with the control group, adiscrepancy significant predominance(P<0.01). Lactating micegroup pPG-2-SLSF/L.casei393and pPG-2-SLSF-co1/L.casei393could detect remarkable specialIgG in the serum of mice, special sIgA level in the stool, vaginal lavage of mice, discrepancypredominance between groups(P<0.05). Compared with the control group, discrepancypredominance(P<0.01). These results indicate that recombinant Lactobacillus casei expressionsystem can stimulate the animal mucosal immune responses and systemic immune responses,specific IgG and sIgA antibody levels of M cell-targeting peptide modified group were significantlyhigher than the unmodified group.The challenge test results showed that one dose immunization after challenge the amount ofdefecation is increase which belong to pPG-2-FaeG/L.casei393group and appears watery stools.There was a mouse in the fourth day of the death, amount of feces gradually reduce start from fifthday and return to normal after the ninth day, protection rate of90%, feces undetectable F4~+ETECstrains after tenth day. pPG-2-FaeG-co1/L.casei393group protection rate is100%, amount of fecesgradually reduce start from the fourth day and return to normal after the ninth day, fecesundetectable F4~+ETEC strains after eigth day. Control mice all died at the second or third day. Thethree immune effects analysis shows that the amount of defecation is increase which belong topPG-2-SLSF/L.casei393group, appears mucous stools at the second day, amount of fecesgradually reduce start from the third day, and return to normal after the sixth day, fecesundetectable F4~+ETEC strains after seventh day. The amount of defecation is mild increase whichbelong to pPG-2-SLSF-co1/L.casei393group and return to normal after the fourth day, fecesundetectable F4~+ETEC strains after fifth day but control mice all died on the second or third dayafter challenged. Lactating mice immune results show that control mice all died on the second day.The amount of defecation is increase which belong to pPG-2-SLSF/L.casei393group afterchallenged, appears mucous stools, amount of defecation gradually reduce start from the fourth dayand return to normal after the severnth day, protection rate is100%, feces undetectable F4~+ETECstrains after eighth day. The amount of defecation is mild increase which belong to pPG-2-SLSF-co1/L.casei393group after challenged, appears mucous stools at the second day,amount of feces gradually reduce start from the fourth day and return to normal after the sixth day,protection rate is100%, feces undetectable F4~+ETEC strains after the seventh day.This study structure prokaryotic expression system of recombinant Lactobacillus caseiexpression the F4~+ETEC immunization protective antigen protein, these results suggest that M celltargeting strategies applied in the system of lactic acid bacteria live vector vaccine is indeedfeasible and the recombinant Lactobacillus casei expression system as an oral vaccine has potentialapplication value. Laid the foundation for the next step to develop the new ETEC oral vaccine, andalso provide basic materials for the exploring targeted transfer antigen enhanced early immuneresponse level of suckling piglets of practice and mechanism research.