| Eimeria tenella,a member of the phylum Apicomplexa,is the causative agent of coccidiosis in chicken,which has long been recognized as an economically significant disease in the poultry industry.To establish and complete endogenous development of the host cell,attachment and invasion of the host cell are key processes.Many proteins have been reported to be involved in these processes,such as microneme proteins(MICs),rhoptry proteins(ROPs),surface antigens(SAGs)and protein kinases(PKs).Recently,some calcium-binding proteins have also been found to be involved in the invasion process of parasites into host cells.The roles of calcium-binding protein of E.tenella in the adhesion and invasion of host cells have not been reported.To control this disease,current strategies rely primarily on prophylactic drugs and attenuated vaccines.However,the rise of drug resistance and the risk of diffusing pathogens have promoted the development of alternative control strategies,such as new vaccines using defined recombinant antigens.Lactobacillus is a common probiotic in the intestine,which can maintain intestinal homeostasis and improve immunity.Genetically engineered lactobacillus can be used as a live vector to present antigen protein,which is an ideal choice for the control of chicken coccidiosis.In this study,monoclonal antibodies against Eimeria tenella membrane associated calcium-binding protein(EtCab)were prepared.EtCab protein was subcellularly localized by indirect immunofluorescence(IFA)in E.tenella sporozoites.The dynamic expression levels of EtCab in different developmental stages of E.tenella were investigated.The function of EtCab in the process of adhesion and invasion was studied.The recombinant Lactobacillus plantarum vaccine expressing EtCab protein on the surface was constructed,and the animal immune protection of the recombinant L.plantarum vaccine was evaluated.The research content is as follows:1.Preparation and application of a monoclonal antibody against the EtCab proteinIn this study,10 hybridoma cell lines stably secreting anti-EtCab m Abs,designated G9D3,D4F10,A2C10,E11C4,G10C8,C5C2,E1C11,G2G7,G6C1 and D9F1,were prepared and generated according to routine procedures,and the antibody subtypes were all Ig G2b,as determined by an ELISA add-on assay in which the E11C4 m Ab recognized one antigen recognition site and the other 9 recognized another.Western blot assays demonstrated that the E11C4 m Ab specifically recognized native EtCab protein,and the titers and subtypes were determined by indirect ELISA,with titers of 1:1.6×10~3in the cell supernatant of E11C4strain and 1:1.28×10~5in purified ascites.An indirect immunofluorescence assay(IFA)using E11C4 m Ab under permeabilized and nonpermeabilized conditions labeled EtCab on the surface of sporozoites.2.The role of EtCab in the invasion of host cellsIn this study,EtCab was cloned and its expression at different developmental stages,adhesive functions and host cell invasion in vitro were investigated.The results of the sequence analysis showed that EtCab contains six EF-hand motifs and the HDEL ER-retention signal belonging to the CREC(45 k Da calcium-binding protein,reticulocalbin,ER calcium-binding protein of 55 k Da,and calumenin)family.IFA using specific polyclonal antibodies under permeabilized and nonpermeabilized conditions labeled EtCab on the surface of sporozoites.Quantitative real-time PCR and western blotting indicated that EtCab was highly transcribed and expressed in sporozoites.The attachment assay using a yeast surface display model showed that the adherence rates of EtCab expressed on the surfaces of yeasts to host cells were 2.5-fold greater than the control.Invasion inhibition assays revealed that specific polyclonal antibodies against EtCab significantly reduced the invasion rate of sporozoites on host cells compared to the control group(P<0.01).These results suggest that EtCab plays an important role in the attachment and invasion of E.tenella to host cells.3.Construction and identification of recombinant L.plantarum vaccineBased on the continuous expression surface display system P32-SP-AP,the signal peptide(SP)and anchor protein(AP)were replaced with Lactobacillus Mur O gene or Bacillus subtilis Pgs A gene,and EtCab gene was inserted.Three recombinant plasmids(P32-Pgs A-EtCab,P32-Mur O-EtCab and P32-SP-EtCab-AP)were constructed and electroporated into L.plantarum NC8.The expression of recombinant EtCab protein was identified by Western blotting.Three recombinant lactobacilli were identified that could surface express recombinant proteins using IFA and their respective surface expression efficiencies were counted.The results showed that three kinds of recombinant L.plantarum could display EtCab protein on the surface.The results showed that the three recombinant Lactobacillus could express EtCab protein.The surface display rate of P32-Pgs A-EtCab-NC8 was the highest(44.90%),that of P32-SP-EtCab-AP-NC8 was 33.87%,and that of P32-Mur O-EtCab-NC8 was 21.25%.The results suggest that all three surface display systems could display exogenous proteins on the surface of L.plantarum,and the P32-Pgs A-EtCab-NC8 had higher protein display efficiency.4.Evaluation of the immunoprotective effect of recombinant L.plantarum vaccineTo study the effect of recombinant L.plantarum vaccine against E.tenella infection,the recombinant L.plantarum P32-Pgs A-EtCab-NC8 with the highest surface display rate was selected for animal immune protection tests.The immune protective effects were evaluated by measuring the average weight gain,cecal lesions,oocyst output,ACI,humoral and cellular immunological functions.The results showed that the ACI of P32-Pgs A-EtCab-NC8 group,NC8 group and PBS group were 170.79,151.45 and 114.77 respectively.The P32-Pgs A-EtCab-NC8 group could ameliorate to some extent the growth retardation caused by infection with E.tenella.The group attenuated the extent of cecal lesions and reduced the expulsion of oocysts.Immunization with P32-Pgs A-EtCab-NC8 could increase the level of Ig G antibody(P<0.05)and the ratio of CD4+/CD8+T lymphocyte subtypes in peripheral blood(P<0.01).In this study,hybridoma cell lines with specificity and stable secretion of Ig G2b monoclonal antibody against EtCab protein were prepared.We have experimentally demonstrated that EtCab is highly expressed in the sporozoite and merozoite stages,localizes on the surface of E.tenella sporozoites and plays a role in the adhesion and the invasion of host cells in vitro.The recombinant L.plantarum vaccine with surface displayed EtCab can effectively prevent chicken coccidia infection. |
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