Preliminary Immunologic Assessment Of Lactobacillus Casei Expressing Transmissible Gastroenteritis Virus S Protein D Site Fusion With Muramyl Dipeptide And Tuftsin

Transmissible gastroenteritis virus (TGEV) is an enteropathogenic coronavirus that resulted ina acute diarrhea in piglets. It’s typically clinical manifestations consisting of severe diarrhea,omitting, and loss water. All kinds ages of pigs are infected. Especially, the mortality of pigletsunder2weeks may reach100%. The more effective methods against TGEV require thedevelopment of novel vaccines that are able to induce mucosal immunization. We have designedLactobacillus casei expressing TGEV protective antigen spike glycoprotein D site with Muramyldipeptide and Tuftsin as recombinant live vaccine, which have both the safety of the traditionalinactive vaccine and the persistence and effectiveness of the vaccine. Because of the Lactobacilluscasei expressing system could not express glycosylated protein as the native expressing system.Themain reason that we choose D site, is that the D antigenic site is continuously sustaining its ownconformation without glycosylation or with glycosylation on a small scale, and D antigenic site ofthe TGEV spike (S) protein is responsible for inducing neutralizing antibody.In this study, Muramyl dipeptide and Tuftsin is being as adjuvants co-expressing with the Dantigenic site. Firstly, synthetizing the gene of Muramyl dipeptide and Tuftsin (20MT) according ofthe bias codons of L.casei, and linkage of20MT for40MT. We use the plasmid pPG612-6Dconnecting the20MT and40MT respectively, and they were transformed into the host strainLactobacllus casei by electrotransformation to generate the recombinant bacteria namedrLc:pPG612-6Ds，rLc:pPG612-20MT-6Ds and rLc:pPG612-40MT-6Ds.The expressed proteins of recombinant strains were induced by2%lactose and analyzed bySDS-PAGE, the results of Western blot analysis indicated27kDa(pPG612-6Ds),43kDa(pPG612-20MT-6Ds) and59kDa（pPG612-40MT-6Ds）which are the special band as weexpected. The secreted protein in the supernatant was analyzed by ELISA in the induced culture.Six to eight week-old BALB/c mice were divided into five groups at randam,12each grouprLc:pPG612-6Ds,rLc:pPG612-20MT-6Ds and rLc:pPG612-40MT-6Ds, group Milk andrLc:pPG612were the negative control which inoculated nothing; the oral groups immunized with live vaccine100μL each mouse every one weeks and immunized continuing3days. Mucosal andSera samples were collected before and after the first immunization of mice, nasal fluids, includingfeces, vaginal mucus, ophthalmic lavage, the intestinal mucus were obtained. The titer of IgG andIgA from mice administered with rLc:pPG612-20MT-6Ds, and rLc:pPG612-40MT-6Ds werehigher than that the group of Lp:pPG612-6Ds, maybe because of the MT. The sera from mice wereable to neutralize TGEV infection on ST cell in vitro with titer rLc:pPG612-6Ds(1:15)，rLc:pPG612-20MT-6Ds(1:32) and rLc:pPG612-40MT-6Ds(1:60).The IgA wererLc:pPG612-6Ds(1:32),rLc:pPG612-20MT-6Ds(1:51) and rLc:pPG612-40MT-6Ds (1:64),respectively.Splenocyte were isolated from mice and seeded at5×106cells/mL, Flow text analysis thesubmit of Th cell, such as Th1,Th2and Th17.in order to understand the function and mechanism ofMT. Meanwhile we test the Treg cell to analysis the effect of immunosuppression. Immunizedgroup of rLc:pPG612-20MT-6Ds and rLc:pPG612-40MT-6Ds secreted Th1,Th2and Th17thatwere significantly higher than negative group of rLc:pPG612. Overall，these data show thatimmunization of recombinant strains rLc:pPG612-20MT-6Ds via oral route produced significantmucous and innate immunization. It suggested that enhanced protection of MT in mucousimmunization is much better than innate type against infection of TGEV.