Sequence Variation In The Toxoplasma Gondii ERP Gene, Establishment Of The ELISA For Detection Of TgERP, And The Studies On Immune Protection Induced By TgERP

Toxoplasma gondii, a zoonotic obligate intracellular parasite distributed worldwide, infects a variety of warm-blooded animals, some birds, and even one-third of the human population, results in serious public health problems and brings economic losses around the world.The first part of the present study aimed to clone the embryogenesis-related protein (ERP) gene of T. gondii and to indicate sequence variation in ERP gene among different T. gondii strains. Genomic DNAs from15T. gondii strains were extracted. Open reading frame (ORF) of the ERP gene was amplified by PCR and sequenced from fifteen T. gondii strains isolated from different geographical locations and hosts. The sequences obtained from this study were aligned using the ClustalX1.83, with the reference sequences of T. gondii ME49strain and VEG strain which were downloaded from http://toxodb.org. Phylogenetic relationships among T. gondii strains from various sources were evaluated using the software Mega5.0and Puzzle5.2. The length of all TgERP sequence was315bp, and the A+T contents were46.67to46.98%. The intra-specific variation among the tested T. gondii strains was0to0.32%. The range of amino acid sequences was0to0.96%. Phylogenetic analysis demonstrated that the TgERP gene can not be used as a marker for analyzing genetic relationships of T. gondii isolates.The second part of the present study amplified the TgERP gene from T. gondii RH genomic DNA by PCR and then cloned the amplicon into the prokaryotic expression plasmid pET-30a (+). The recombinant plasmid p30a-ERP was transformed into Escherichia coli BL21(DE3) and induced by IPTG. The expressed product was purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by SDS-PAGE. The polyclonal antibody against the recombinant protein was prepared by immunizing New Zealand white rabbits with the purified protein. PCR amplification and restriction analysis proved that the restriction prokaryotic expression plasmid pET-30a (+) was correctly constructed and sequencing results showed that the cloned TgERP gene was identical to the reference sequence reported (http://toxodb.org). The TgERP protein was expressed under1.0mmol/L IPTG at37℃, shaken for6h. SDS-PAGE analysis showed the protein product was successfully purified with a molecular weight of about16.7ku. Western-blotting and ELISA analyses indicated that the TgERP protein reacted with sera from vaccinated rabbit, indicating that it could be a potential candidate antigen for developing new detection methods or new sub-unit vaccine against toxoplasmosis. Then by using the recombinant protein as coated antigen and HRP-SPA as second antibody, an indirect ELISA for detection of anti-T.gondii antibody was established. ELISA results showed that reaction condition was best when antigen concentration was2.5μg/mL, primary antibody dilution was1:200, and blocked by incubation for0.5h with a2.5%(w/v) solution of skimmed milk powder in PBST, the antiserum and test samples were reacted first for15minutes as the resulting mixture, secondary antibody dilution was1:10000and incubated for30minutes, colour development for10minutes. The result showed that the infection rate, infected through oocyst, is0.664%.The third part of the present study constructed subunit vaccine based on TgERP gene. The subunit vaccine were diluted to5μg/μL, and100μL were subcutaneously injected into the Kunming mice Group IV, whereas group Ⅰ to group Ⅲ were injected with PBS, PBS+206adjuvant, and nothing as controls, respectively. Two booster injections were given at the2nd week and the4th week after the first injection. Levels of IgG, IgGl and IgG2a antibody, the cytokines of IFN-y, IL-2, IL-4, IL-10, IL12p70, and CD3+CD4+CD8-and CD3+CD8+CD4-cell were examined. The result showed that the levels of IgG, IgG1, and IgG2a antibody are higher than the control groups, the cytokines of IFN-y, IL-2, and IL12p70are higher than the control groups too, IL-4is lower compared with the control groups. And content of CD3+CD8+CD4-and CD3+CD4+CD8-cell has significant increase. So we conclude that the subunit vaccine is suitable for clinical infection experiments.In summary, phylogenetic analysis exhibited that TgERP gene probably can not serve as a marker for studying genetic relationships of various T. gondii isolates. An indirect ELISA for detection of anti-T.gondii antibody was established. And its efficacy as a subunit vaccine was evaluated. The research on the ELISA and vaccine presented satisfied results.