Expression Of The Different Antigen Fragments Of The Major Structural Protein Of Porcine Epidemic Diarrhea Virus In Recombinant Lactobacillus Casei And Immunology Evaluation

Porcine epidemic diarrhea virus(PEDV),a member of the Coronaviridae,causes an economically important disease in neonatal piglets,which characterized by severe enteritis with anorexia,vomiting,acute diarrhea,dehydration and significant mortality in swine,thereby incurring serious economic losses worldwide due to the death of neonatal piglets and weight loss of the infected pigs.Morbidity and mortality in infected neonatal piglets less than 5 days old approach 100%because of severe diarrhea and dehydration.However,mortality in infected piglets older than 10 days is less than 10%.PED have been reported in many countries,caused serious economic losses due to the death of neonatal piglets and weight loss of the infected pigs.Lactic acid bacteria(LAB),considered to be safe bacteria with a GRAS(generally regarded as safe) status,possesses many properties that has been thought to be an ideal vaccine which has a good perspective.The main goals of this thesis were:(1) to understand the knowledge of the data of molecular biology of PEDV strains that are prevalent in China;(2) and select the protective antigen of PEDV; (3) to study the efficacy,immunogenicity of recombinant Lactobacillus casei after oral vaccination.Above all,we cloned s gene of PEDV LJB/03 strain,then the gene fragment was subcloned into pMD18-T vector,sequenced and analysised.Comparing with PEDV reference strains,S gene had some mutant,and homology of nucleotides and amino acids sequences was between 94.4%and 100%.The LJB/03 n gene has also a high sequence homology to those of other PEDV isolates.The analysis result showed that LJB/03 have high homology with different isolates,so it can serve as target antigen to develop genetic engineering vaccine.Some of the structural proteins of PEDV carry major epitopes involved in virus neutralization and are essential for the induction of protective humoral responses and the development of an effective vaccine.Rabbit antisera were prepared using full-length N proteins and five truncated expressed fragments covering the S protein fragments[s1(22-505aa),S2(488-980aa), S34(951-1383aa),PS420(461-638aa),S22(502-792aa)].Antisera to S proteins were found to have different neutralizing titres towards PEDV infection in vivo,ranging from 1:23～1:93.Antiserum to the N protein did not contain neutralizing antibodies.Epitopes inducing protective humoral responses to virus infection were located mainly in the region S1,S22,PS420.The in vitro neutralization assay has important implications for the design of an effective vaccine preventing PEDV infection.The selective gene ps1(including S1,S22,PS420),ps420,n,1p0(LTB and ps420) were subcloned into the expression vector pPG-1 or pPG-2,and then transformed into L.casei 393 by electroporation,resulting in recombinant strain pPG-1-n/L.casei393,pPG-1-ps1/L.casei393,pPG-1 -1p0/L.casei393,pPG-1-ps420/L.casei393,pPG-2-n/L.casei393,pPG-2-ps1/L.casei393,pPG-2-1p0-/L.casei393, pPG-2-ps420/L.casei393,respectively.The recombinant strains constructed in this study were induced by 2%lactose in MRS medium to express interest protein.The expression protein in the cell-surface expression system (pPG-1-n/L.casei393,pPG-1-ps1/L.casei393,pPG-1-1p0/L.casei393,pPG-1-ps420/L.casei393) was detected by Western blot and the whole bacterica ELISA.The indirect immunofluorescence test showed that the expressed protein was displayed on the cell surface of L.casei 393.The recombinant strain pPG-2-n/L.casei393,pPG-2-psl/L.casei393,pPG-2-1p0/L.casei393, pPG-2-ps420/L.casei393 was detected via Western blot and indirect ELISA.The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein.The indirect ELISA test also indicated that the interest protein was expressed and secreted into the culture supernatant.To evaluate the immune responses of recombinant L.casei 393 expressing PEDV protein as oral vaccine,BALB/C mouse were used as animal model immunized with recombinant strains by intragastric administration,and the immune efficacy was analyzed.The immune protocol was administered on three consecutive days at days 0,1and 2.A booster immunization was given at days 14,15 and 16 and a second booster was given at days 28,29 and 30,the mice were fed with 10~9 recombinant strains.The groups include pPG-1-1p0/L.casei393 immun-ization group,pPG-2-1p0/L.casei393 immunization group,pPG-1-ps420/L.casei393 immunization group,pPG-2-ps420/L.casei393 immunization group,pPG-1-n/L.casei393 imm -unization group,pPG-2-n/L.casei393 immunization group,pPG-1-ps1/L.casei393 immunization group,pPG-2-ps1/L.casei-393 immunization group,pPG1 divalent immunization group(pPG-1-n /L.casei393 and pPG-1-ps1/L.casei393 ),pPG2 divalent immunization group(pPG-2-n/L.casei393 and pPG-2-ps1/L.casei393).As control,the mice were immunized with L.casei393 harboring pPG-2 or pPG-2,or PBS by oral route.Specific anti-PEDV protein sIgA was detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces,nasal wash,vaginal lavage,eye wash,intestines mucus of mice after intragastric administration,and Specific IgG was detected by indirect ELISA in the serum of immunized mice.The spleen lymphocyte proliferation index(LPI) was measured.IFN-γand IL-4 contents in the supematant of spleen cell culture were also assessed by ELISA.The results of ELISA showed that the mice immunized with recombinant strains could produce remarkable special slgA level in the feces,nasal wash,vaginal lavage,eye wash of mice on days 21 post oral administration,and highest level were obtained on days 35 or 49 post oral administration. Highest level of anti-PEDV protein IgG in the serum of mice were obtained on days 35 or 49 post oral administration.Special sIgA level in the intestines lavages significant increase.These indicated that the recombinant strains constructed in this study could induce both mucosal immune and system immune responses.The results also demonstrated that recombinant L.casei 393 of cell surface expression type could elicit higher immune response level than that induced by secretion expression type.The multiple oral immunizations with recombinant L.casei 393 could induce significantly specific mucosal IgA response as well as serum IgG responses.Moreover,there was significant increase of IFN-γand IL-4 contents in the supernatant of spleen cell culture in immunized group. Statistical significant difference was observed among the LPI among these ten immunized groups of mice and control groups.The results demonstrated that recombinant L.casei 393 of secretion expression type could elicit higher immune response level than that induced by cell surface expression type.pPG-1 divalent immunization group(pPG-1-n/L.casei393 and pPG-1-ps1 -/L.casei393),pPG-1 divalent immunization group(pPG-2-n/L.casei393 and pPG-2-ps1 -/L.casei393 ) can induce more effective immune response,leads to synergic effect.The induced antibodies in serum and intestinal lavages demonstrated neutralizing effect on PEDV infection.Another conclusion is LTB could be an effective advjant in intragastric administeration of recombinant L.casei 393.Our data has indicated that intragastric administration of the recombinant L.casei 393 could induce specific immune response against PEDV.Our work established a good foundation for further study on the new and effective PEDV recombinant oral vaccines.