| Avian influenza(AI)caused by Avian Influenza virus(AIV)is an acute and highly contagious disease,which generates serious economic losses to poultry farming and threatening human health.So far,vaccine is the most effective way to control AI.Considering the variability of avian influenza virus,it’s an urgent need to develop more convenient and effective vaccine to solve this problem.Lactobacillus plantarum is a good characteristic of heterologous protein delivery system,which could have the effect of prevention and treatment of disease in certain extent by oral recombinant Lactobacillus plantarum.Poly-γ-Glutamic Acid Synthetase A(pgsA)can stably anchor the exogenous proteins on cell surface.Recombinant strains with pgsA’ protein takes the advantage of expression quantity of heterologous protein and the performance of the antagonism to inflammation reactivity of cells.Dendritic cells targeting peptide(DCpep)which is beneficial to mucosal immune response plays a crucial role in the mucosal immune homeostasis.Heat labile toxin B subunit(LTB)from E.coli is an effective vaccine adjuvant.In this study we chose Lactobacillus plantarumt as a delivery vector of exogenous proteins to construct a new functional lactic acid bacteria with fusing the pgsA’ surface display element with the highly conserved protein HA2 gene of AIV,together with the immune adjuvant LTB peptide sequence or immune induction DCpep.And then we evaluated and analyzed the immune effect in mice.Theprimary procedures and outcomes of the study are described as follows:(1)Construction of novel recombinant lactic acid bacteria and expression of the targeted proreinsThe recombinant plasmids pSIP409-pgsA’-HA2,pSIP409-pgsA’-HA2-DCpep and pSIP409-pgsA’-HA2-LTB wereconstructed,then electroporated into Lactobacillus plantarum.We determined the size and immunogenicity of the expressed proteins by western blotting approaches,after the induction by induced peptide.We also determined the anchoring of expressed protein by fluorescence on the surface of Lactobacillus plantarum by immunofluorescence(IF)testing.Results showed that: The new functional lactobacillus pSIP409-pgsA’-HA2、pSIP409-pgsA’-HA2-DCpep and pSIP409-pgsA’-HA2-LTB were successfully constructed and the immunogenicity of the recombinant strains were verified by western blotting.And intense fluorescence signals were detected by IF assay to prove that HA2 was anchored on cell surface for certain.The results obtained set upa stable foundation for the further investigation.(2)Detection of Molecular immune mechanism effect to the new functional lactic acid bacteriaMice were inoculated with the novel functional lactic acid bacteria according to immunization protocol.The activation of T and B cells in related immune organs of the mice were determined by flow cytometry.The HA2 specific IgG and SIgA in vivo and cytokines production in serum were measured by ELISA.Mice challenged with AIV were then monitored for two weeksto calculate body weight loss and survival rates.And the lung pathological section was determined.The results showed that: the new functional lactic acid bacteria effectivelypromoted the activation of CD3+ CD8+Tlymphocytes in spleen and MLNs,the levels of CD3+ CD4+IL4+,CD3+ CD4+IL17+ and CD3+ CD4+IFN-γ+ T cells,as well as CD3+ CD8+IFN-γ+ T cells.In addition,the B220+ IgA+B lymphocytesand HA specificIgG,SIgA and cytokines were also increased.After challenged with AIV,the mice in HA2-Dcpep 、HA2-LTB immunized group achieved the lowest body weights at day 7 and then recovered after that,while as the mice in other groups achieved the lowest body weights at day 8,then recovered until last.The survival rates were recored as 12.5%,12.5%,12.5%,25% and 37% in mice treated with control,pSIP409-pgsA’,pSIP409-pgsA’-HA2,pSIP409-pgsA’-HA2-Dcpep and pSIP409-pgsA’-HA2-LTB,respectively.The lung pathological section showed that: the new functional lactic acid bacteria could alleviate inflammation of the lungs in mice challenged with AIV,reducing pathological damage in the lungs,and prevent infection of AIV in some certain.The new functional lactic acid bacteria with immune molecule DCpep has a significantly effective,but the strain with adjuvant molecule of LTB showed more immune effect to H9N2 than DCpep.Conclusions show that: the construction and expression of the new functional lactic acid bacteria are successful.As an anchor gene,pgsA’ could stably anchor protein on the cell surface of Lactobacillus plantarumto promote the role of the protein in the extracellular spaces,stimulating the secretion of specific IgG and SIgA,enhancing the activation of T lymphocyte and B lymphocyte,improving the levels of humoral and cellular immunity.This study provides a basis for the development of anti-avian flu oral probiotics... |
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