Protective Eiffcacy Induced By Recombinant Proteins And DNA Vaccine Encoding A Repair_PSⅡ Gene Against Eimeria Tenella Challenge

Coccidiosis is caused by species of intracellular protozoan parasites belongingto the genus. E.tenella is one of the most important species associated withcoccidiosis in chickens. Current means of control rely on the use ofchemotherapeutics and live vaccines. However, these are associated with irreversibledrawbacks like drug resistance, drug residues, live vaccines scttered poison, etc. Thecoccidiosis recombinant vaccine is still current hot spots and trends of theimmunization of chicken coccidia, but the protective effect of such vaccines are notyet ideal. Therefore, we cloned a new coccidiosis vaccine candidate genes, and thenuse the gene to prepare subunit vaccines and DNA vaccine, and finally investigatechicken body’s immune response stimulated by the vaccine and protective effectagainst E.tenella, which is of great significance for the development and research ofE.tenella recombinant vaccine.Gene cloning and prokaryotic expression of Eimeria tenella Repair_PSII: Wescreened a new gene from E.tenella sporulated oocysts cDNA library, containing anopen reading frame of816bp which encode271amino acids. Repair_PSII protein is atransmembrane protein with unknown function. It has a conserve domain ofRepair_PSII family. In this study, the recombinant Repair_PSII protein wassuccessfully expressed using prokaryotic expression vector in the soluble form. Also,the recombinant Repair_PSII protein can be identified by anti-E.tenella oocystspolyclonal antibody. IFA showed Repair_PSII protein expressed in the sporozoitestage.The protective effect of the recombinant Repair_PSII protein against Eimeriatenella challenge: Chicks were immunized with the recombinant Repair_PSII proteinin different immune pathways, and the immune response and protective effect againstE.tenella challenge elicited by this protein were evaluated. The results showed thatsubcutaneous immunization and taking engineered bacteria orally elicited specificantibody and cell-mediated immune response (the levels of IL-2and IFN_-γ, thepercentages of CD4⁺and CD8⁺Tlymphocyte cells, T lymphocyte stimulation indexwere significant difference compared with control groups). Challenge experiments demonstrated that subcutaneous immunization and taking engineered bacteria orallycould reduce the numbers of oocysts, decrease cecal lesion and increase bodyweightgains. The comprehensive evaluation of ACI values were173.2and165.6respectively.The construction of eukaryotic plasmid pVAX1-Repair_PSII and protectiveeffect against Eimeria tenella challenge: the Repair_PSII gene of E. tenella wassubcloned pVAX1and protein was expressed in Hela cells. The results showed thatthe pVAX1-Repair_PSII eukaryotic plasmid was successfully constructed. Therecombinant protein was expressed in Hela cells and verified by IFA, SDS-PAGE andWestern blot analysis. Chickens were immunized with the recombinant plasmidpVAX1-Repair_PSII and the immune response and protective effect against E.tenellachallenge elicited by this protein were evaluated. The results showed that theRepair_PSII DNA vaccine was capable of eliciting specific antibody andcell-mediated immune response (the levels of IL-2and IFN_-γ, the percentages ofCD4+and CD8+T lymphocyte cells, T lymphocyte stimulation index were significantdifference compared with control groups). Challenge experiments demonstrated thatthe immunization of the Repair_PSII DNA vaccine can reduce the numbers of oocysts,alleviat cecal lesion and body weight loss. The comprehensive evaluation of ACIvalue was178.7.In this study, Repair_PSII as a new candidate for E.tenella vaccine was cloned.Good protective efficacy was induced by recombinant proteins and DNA vaccineencoding a Repair_PSII gene against E.tenella challenge. Which has an importanttheoretical significance and application prospects on immunologic prevention ofcoccidiosis.