Construction And Immunogenicity Evaluation Of Recombinant Lactobacillus Casei Expressing BVDV E2 Protein And Development Of An Antigen Capture ELISA For BVDV-1 Detection

Bovine viral diarrhea(BVD)is one of the most significant infectious endemic diseases in world-wide cattle population and causes substantial economic losses due to its detrimental effect on production and general health conditions.Bovine viral diarrhea virus(BVDV)is an etiological agent of BVD belonging to the Pestivirus genus within the Flaviviridae family.Due to its extreme complex pathogenesis and immunosuppression characteristics of BVDV,failure of controlling the disease are often reported.Proper vaccination schedule along with accurate detection and elimination of persistently infected aninal is necessary for the effective and sustainable BVDV control strategy.In this study,we have constructed and evaluated the imnunogenicity of a recombinant Lactobacillus casei(L.casei)expressing BVDV E2 protein,discover the anti-BVDV activity of L.casei and developed an antigen-capture-ELISA for the detection of BVDV,aiming to provide the technological support for this disease prevention and control.1.Immunogenicity evaluation of recombinant L.casei expressing BVDV E2 protein1.1 Construction of recombinant L.casei expressing BVDV envelop-glycoprotein E2 BVDV E2 protein was reported to induce highly neutralizing imnune response against infection and Lactobacillus strains has been used in the practice of diarrhea management and also as a heterologous gene-expressing vector.Firstly,the E2 gene of BVDV-1(NADL-AV67 strain)virus was amplified by RT-PCR and cloned into pELXI,a growth phase-dependent surface expression system,to construct recombinant vector pELX1-E2.Transformation into BL-21 competent cells for protein expression was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting(WB)analysis.The recombinant vector was further electroporated into L.casei competent cells to yield recombinant L.casei/nELX1-E2 strain.The expression by the recombinant L.casei strain was verified by SDS-PAGE,WB and a considerable amount of E2 protein with-40kDa expression was found best when the OD600 of the growth media reached at 1.5.Immunofluorescence microscopic analysis indicated the surface expression of E2.1.2 The immunogenicity evaluation of the L.casei/pELX1-E2 in BALB/c miceThree groups of mice were immunized with the equal amount(1010 CFU/mL)of L.casei/pELX1-E2 through oral,intranasal(IN)and intramuscular(IM)routes and another two control groups of mice were administered with L.casei/pELX1(Cont.L.casei)orally and sterile PBS orally(Cont.PBS)respectively.Boost vaccinations were performed for three times after two weeks with the same dose and route.For serological study(IgG,IFN-?,and IL-12)sera were collected on 0,14,28 and 42 day post immunization(dpi)via tail vein punching.Feces and intestine were collected to detect local immune response(IgA).The rest of the mice were challenged with BVDV-1 at a dose of 10-5 TCID50 to judge the protective efficacy.The result revealed that oral and IN administrations of the recombinant strain could induce a significantly higher level(p ? 0.01)of specific anti-E2 mucosal IgA and serum IgG as well as the greater level of cellular response indicated by IFN-? and IL-12 than those of IM groups of mice.However,IN vaccination was found the most potent approach to elicit the local,cellular and systemic immune responses than other immunization groups.The serum neutralizing antibody titer of 1:128 and stronger protection efficacy against BVDV could be induced by L.casei/pELX1-E2.Therefore,this recombinant L.casei expressing E2 could be a safe and promising mucosal vaccine candidate against BVD.2.The anti-BVDV activity of L.casei MCJ protein based metabolites(LPM)Lactobacillus spp.has widely been using in the field of management and treatment of gastro-enteric disease for both human and animal.The ability of LPM to suppress BVDV infection in Madin-Darby Bovine Kidney(MDBK)cell line was demonstrated in this study.The protein-based metabolites were extracted from the cultured L.casezi to obtain the safest and beneficial form of the probiotic bacteria.The results reveal that LPM have no cytotoxic effect and the cell viability remain more than 80%even after the cells are treated with 3000(ig/mL of LPM.The results of plaque formation unit(pfu)assay showed that LPM can reduce the viral infection rate.To know the mechanism of LPM for anti-BVDV activity,MDBK cells were exposed to LPM before,after and co-incubation of virus infection.The co-treatment of LPM with BVDV revealed the best results for the decreased viral protein expression by Western blot,RNA copy number by Quantitative real-time PCR and fewer BVDV infected cells by Immunofluorescences assay.The results suggest that the LPM has a potential anti-BVDV activity which could be a prospective application for the prevention and control of BVDV infection.3.Development of ELISA for detection of BVDV-13.1 Recombinant protein expression and monoelonal/potyclonal antibody production The NS3 gene of BVDV-1 was amplified cloned into pET-30a(+)plasmid,expressed in E.colI.The expressed?57 kDa NS3 protein and?40 kDa E2 protein from previous construction were purified and used for the further experiment.Murine monoclonal antibodies(MAb)were prepared against E2 protein and produced in group of 4 week old BALB/c mice(n = 5).The positive stable hybridoma panels secreting anti-E2 MAb(IB-10)were selected with the highest reactivity against both rE2 and BVDV-1 antigens respectively,through indirect-ELISA.The antibody titers of IB-10 clones were found 1:12,800.The hybridoma cells were used to immunize mice for ascites production.Polyclonal antibodies(PAb)were produced against the recombinant NS3 protein in 10 week old New Zealand white rabbit.Production of MAb and PAb were confirmed through indirect ELISA,WB,and IFA analysis3.2 Optimization of antigen-capture ELISACheckerboard titration method was used for determination of reaction conditions to select the highest P/N value.The rabbit anti-rNS3 as a capture Pab and mice anti-E2 as detector MAb were used to develop this AC-ELISA for BVDV-1 detection.The cut-off value was calculated by means of 25 BVDV negative white blood cells prepared from whole blood samples and fixed at 0.523.The detection limit of this assay was found 50 ng/mL of total BVDV protein and 103 TCID50 of BVDV.The average intra and inter assay coefficient variation was found 3.87%and 6.90%respectively.This AC-ELISA could differentiate BVDV effectively from other bovine and porcine diarrhea-causing organisms without any false positive results.3.3 Detection of artificial samplesBVDV was artificially added into leukocyte samples,skin and lung samples.The Kappa values were measured simultaneously by this method,TaqMan qRT PCR and traditional RT-PCR.The Kappa values between the method and the two methods are 0.86,1;0.86,0.72 and 0.72,0.85 consecutively which revealed highly agreement.It shows that a sensitive and specific BVDV-1 antigen capture ELISA method has been established,butneed further clinical BVDV evaluation.In conclusion,the immunogenicity of the recombinant L.casei expressing BVDV E2 protein was evaluated in mice model and showed potential novel vaccine against BVDV infection.L.casei was found with anti-BVDV activity in vitro further indicating the new therapeutical function of L.casei in prevention of the BVDV infection.Finally,the antigen-capture ELISA with high sensitivity and specificity has been developed providing a low-cost method to detect BVDV.The results of this research provided the useful tools for BVDV prevention and control.