Construction Of An Engineering Strain Expressing Rabies Virus Glycoprotein Lactic Acid Bacteria And Oral Immunization

Rabies is an acute infectious disease caused by Rabies virus(RABV).It is common in humans and animals.Once it develops,the mortality rate is almost 100%.The most effective means of controlling human rabies infection is to cut off the source of infection and control the spread of rabies among animals.The difficulty in prevention and control lies in the immunity of stray animals and wild animals.Therefore,there is an urgent need to develop an oral vaccine suitable for large-scale vaccination that does not rely on injection vaccination.Lactic acid bacteria are recognized as safe probiotics and have good tolerance to the adverse environment of the intestine,and proteins expressed by recombinant lactic acid bacteria can be produced on a large scale without purification.In this study,lactic acid bacteria was used as the expression vector,and rabies virus glycoprotein(GP)was used as a candidate antigen to construct a recombinant lactic acid bacteria expressing rabies virus glycoprotein as a new oral vaccine.It is evaluated as a new oral vaccine by in vitro and in vivo tests to provide a new strategy for the development of rabies oral vaccine.1.According to the rabies virus gene sequence contained in Genbank,the main antigen epitope region gene RVG of rabies virus glycoprotein is synthesized.The specific dendritic cell targeting peptide(DCpep)-encoding gene was fused at its C’ end by PCR and inserted into the lactic acid bacteria expression vector,and the fusion-independent peptide(ctrl)-encoding gene was used as a control.Recombinant lactic acid bacteria NC8-pgsA’-RVG-DCpep and NC8-pgsA’-RVG-ctrl expressing the RVG gene were separately constructed.Protein level detection by Western-blot and immunofluorescence showed that the rabies virus glycoprotein was successfully expressed on the surface of lactic acid bacteria.The recombinant lactic acid bacteria were orally immunized to mice,and their immunogenicity was evaluated,and detection was carried out by flow cytometry and ELISA.The results showed that both recombinant lactic acid bacteria were immunogenic,but the recombinant lactic acid bacteria NC8-pgsA’-RVG-DCpep could induce the activation of more CD11c+CD80+DC cells compared with the recombinant lactic acid bacteria NC8-pgsA’-RVG-ctrl.And B220 + IgA + B cells,increase the amount of sIgA and serum IgG secretion in the intestine.It was shown that the recombinant lactic acid bacteria NC8-pgsA’-RVG-DCpep was more immunogenic.2.In this study,two recombinant lactic acid bacteria containing double-copy glycoprotein(GP)gene were constructed,which were named as NC8-pSIP-dRVG and NC8-pgsa-dRVG,in order to increase the expression of antigenic protein.To achieve the purpose of improving the immune effect.The results of Western-blot and immunofluorescence showed that compared with the recombinant lactic acid bacteria containing single copy GP gene,the recombinant lactic acid bacteria containing double copy GP gene produced higher glycoprotein,and the intracellular expression of recombinant lactic acid bacteria NC8 containing double copy GP gene.The amount of GP protein expressed by pSIP-dRVG was significantly higher than that of recombinant lactic acid bacteria NC8-pgsa-dRVG containing double copy GP gene.3.The mice were orally immunized to evaluate the immunoprotective effect of the double-copy GP recombinant lactic acid bacteria.In the mouse immunization experiment,recombinant lactic acid bacteria containing single copy GP gene and recombinant lactic acid bacteria containing double copy were immunogenic,but recombinant lactic acid bacteria containing double copy GP gene could induce more production than recombinant lactic acid bacteria containing single copy GP gene.Glycoprotein-specific antibodies,IgG and sIgA,and increase the secretion of cytokines IFN-γ,IL-17 A,IL-4 in serum;recombinant lactic acid bacteria containing double copy GP gene can activate or recruit more dendrites Dendritic cells(DCs),and induced a significant increase in CD4+ T and CD8+ T cells secreting IFN-γ or IL-4.The recombinant effect of the recombinant lactic acid bacteria NC8-pSIP-dRVG containing the double-copy GP gene was better than that of the surface-displayed double-copy GP gene recombinant lactic acid bacteria NC8-pgsa-dRVG.Under the lethal challenge of RABV,the protective rate of intracellular expression of recombinant lactic acid bacteria NC8-pSIP-dRVG containing double copy GP gene was significantly higher than that of surface-displayed recombinant lactic acid bacteria NC8-pgsa-dRVG containing double copy GP gene.60%,equivalent to a single immunization protection rate for commercial inactivated vaccines.Therefore,the intracellular expression of recombinant lactic acid bacteria NC8-pSIP-dRVG containing double copy GP gene has the potential to become a new rabies oral vaccine,and provides a new strategy for the development of rabies virus oral vaccine.