TGEV S Protein Expressed In Recombinant Targeting Bacillus Subtilis To Enhance The Mucosal Immunity In Pigs

Bacillus subtilis(B.subtilis)is a kind of common probiotics,widely used as antigen delivery vector and mucosal immune enhancer,and can induce protective immune response.Transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PEDV)cause severe diarrhea,vomiting and dehydration in piglets,resulting in huge economic losses to the pig industry.In order to develop an effective,safe,and convenient oral vaccines.In this study,we constructed a recombinant Bacillus subtilis strain(B.subtilis CotGSG)displaying the TGEV spike(S)protein on the spore's suface.Secondly,the effect of local mucosal and systemic immune response was detected after oral immunizated with B.subtilis CotGSG in piglets.Thirdly,discussed immune function of B,subtilis CotGSG to intestinal submucosal dendritic cells(DCs)with the piglets intestinal ligated loop model in vivo and bone marrow-derived dendritic cells in vitro.Finally,explored to enhance the immune effect of B.subtilis through the intestinal M cells.This study is divided into the following four parts:1 Construction of recombinant B.subtilis CotGSGIn this experiment,firstly,amyE 5' end,amyE 3' end and Kan fragments were amplified from B.subtilis WB800 genome,Kanamycin resistant gene was amplified from pMK3 plasmid.Three fragments were inserted into the vector pCDNA3.1(+)by T4 DNA ligase to generate the integration vector pInAmyE.Then spore coat protein CotG(involved promoter),TGEV S gene and GFP fragments were overlaped together and inserted into pInAmyE by the site of Hind? to generate pInCotGSG.Based on the stable transformation condition,the pInCotGSG plasmid was transformed into B.subtilis WB800 strain to obtain recombinant B.subtilis CotGSG.Analyse the TGEV S gene in the genome of B.subtilis WB800 by PCR.And detection the expression and surface display of fusion protein.The results showed TGEV S protein could be expressed on the surface of B.subtilis CotGSG by western-blot and confocal microscopy.2 The immune effect of B.subtilis CotGSG on piglets orallyThis experiment based on the B.subtilis CotGSG obtained by transformating in the first chapter.Firstly one-month-old piglets were orally administered and then detected the local mucosal immune effect including specific SIgA antibody and lymphocytes levels.Secondly,explored the influence of serum specific IgG and neutralizing antibody levels on the system immune.The results found that B.subtilis CotGSG spores,could induced TGEV specific SIgA antibody by oral immunization piglets after 7 days,and could significantly increase the number of intestinal intraepithelial lymphocytes(IELs)and CD3+ T lymphocytes after 35 days.These results suggested that B.subtilis CotGSG spores could induce intestinal mucosal immune response.In the detection of systemic immune responses,we found that B.subtilis CotGSG spores could increase high level serum TGEV specific IgG antibody and neutralizing antibody titers after oral immunization piglets,and was significantly higher than inactivated TGEV group in the middle.From the results above,after oral administrated piglets,the expression of TGEV S protein of recombinant Bacillus subtilis could induce humoral and cellular TGEV specific immune responses,and to a certain extent,protect the TGEV infection.The research aimed to study the prevention of TGE live vector vaccines research.3 The mechanism of B.subtilis induced mucosal immunityDCs mediated mucosal immune response depends on the uptake of DCs on antigen and its ability to mature.Uptake microbial antigens is the first step to start the mucosal immunity of DCs.After processing antigens,DCs present the antigens to subcutaneous lymphocytes or migrate to nearby lymph tissue,further activate the initial T,B lymphocytes.Firstly,observe the ability of DCs uptake B.subtilis spores in piglets intestinal ligated loop model,DCs carrying B.subtilis spores migrate to nearby lymph node.Secondly,isolated and cultured bone marrow-derived dendritic cells of piglet in vitro,and the effects of B.subtilis spores on bone marrow dendritic cells mature ability and further to induce proliferation of T cells.The results showed DCs could effectively uptake B.subtilis spores and further migrate to the mesenteric lymph nodes by confocal microscopy and flow cytometry.In addition,B.subtilis spores could also promote the intestinal CCL20 mRNA expression level.B.subtilis spores could stimulate DCs maturation and further promote the proliferation and differentiation of T cells.This showed that the B.subtilis CotGSG stimulated the secretion of CCL20 and recruited DCs to the intestinal mucosa,after DCs uptaking and processing the B.subtilis CotGSG spores,migrating to the mesenteric lymph nodes,and promoting T cell proliferation,which might be an important mechanism of effective specific immune responses induced by B.subtilis CotGSG spores.4 Enhance the effect of B.subtilis mucosal immunityM cells is a kind of specialized intestinal epithelial cells,which can effectively absorb the antigen and present to the lower DCs,macrophages,T/B lymphocytes and other immune cells,and further induce specific immune responses.In this study,in order to enhance the delivery effect of B.subtilis.First of all,constructed the recombinant B.subtilis(B.subtilis RCL)expressing the targeting M cells protein(L-Lectin-?-GF)and PEDV S protein.Secondly,analyse the co-localization of M cells and B.subtilis RCL by intestinal ligated loop model.Then analyse the specific SIgA antibody levels in the intestinal washing fluid and specific IgG antibody level in serum orally immunized mice.The results showed that orally administrated B.subtilis RCL could effectively induce thenumber of M cells and the increase of non-specific SIgA antibody level in the intestine.B.subtilis RCL could co-localization with M cells the intestinal,and after a period of time,B.subtilis RCL could effectively enter the lymphatic tissue.After oral immunization of mice,B.subtilis RCL could induce significant intestinal PEDV specific SIgA antibody and serum IgG antibody.According to the results above,B.subtilis RCL could enhance the intestinal mucosal immune response and induce systemic immune response through M cells.The results provide a theoretical basis for enhancing the delivery efficiency of Bacillus subtilis on mucosal immune.