Screening,Identification And Functional Study Of Differential Antigens Of Eimeria Tenella

Chicken coccidiosis is one of the most serious parasitic disease that endangers the chicken industry and brings huge economic losses every year.Chicken coccidiosis prevention and control is facing severe challenges,such as emergence of resistant strains,drug residues,costly production,and emergence of toxins.With the development of modern biotechnology,the third-generation vaccine is considered to be an ideal anticoccidial vaccine.Screening and identification of protection and specific antigens with strong immunogenicity is the basis and key to development of third-generation vaccines.In the present study,the specific antigen group of E.tenella were screened by comparative immunoproteomics.E.tenella specific antigens were identified by immunogenicity experiments and the functions of specific antigens were studied.It provides theoretical basis and new ideas for the development of the subunit vaccine of chicken coccidia.The research includes the following six aspects:1.Preparation and identification High-titer immune serum of E.tenella and E.maximaIn the present study,the Eimeria high-titer immune serum were prepared by multiple oral infection of chicken coccidia oocysts.The quality of high-titer immune serum were identified by ELISA,Western blot,in vitro blocking and passive immunity assay.ELISA results showed that the serum antibody increased with the increase of the number of infection,and reached a peak at 8 dpi after the fifth infection.IFA results show that the antigens are evenly distributed on the cell membrane and cytoplasm of the parasite.The merozoites proteins were analysed by Western blot using the high-titer immune serum as the primary antibody.The results showed that the number and response intensity recognized by E.tenella immune sera were higher than E.maxima immune sera.The antibody blocking assay showed that the invasion rate of sporozoite incubated with E.tenella immune sera was significant lower than that incubated with E.maxima immune sera.Passive immunity experiments show that E.tenella immune serum can provide good immune protection?ACI=181.26?,while E.maxima immune serum can only provide limited immune protection?ACI=141.36?.These results show that there are significant differences in the E.maxima and E.tenella serum antibodies that from the aspects of antigen-antibody reaction and difference of antigenic,which can be used for immunoproteomics analysis.2.Screening of the E.tenella specific antigensIn order to systematically screen the specific antigen group of E.tenella,the E.tenella merozoites protein were analysed by immunoproteome.The merozoite proteins of E.tenella were analysed by Western blot,using the E.maxima and E.tenella high-titer immune serum as primary antibodies.The immunohybrid protein spots were analysed by PD Quest.The results showed that 109 significantly different protein spots were identified.The points more than 2.0 times were identified by mass spectrometry analysis,and 36 antigen genes were identified successfully.To verify the reliability of immunoproteome,EtHSP60,EtSERPIN1,Etprofilin,EtCCT2,Etpyruvate kinase,EtMIC1,EtRACK,and EtG-3-P were selected for prokaryotic expression.The recombinant proteins were analysed by Western blot,using E.maxima and E.tenella high-titer immune serum as primary antibodies.The results showed that the coincidence rate with the results of immunoproteomics was 83.33%.3.Preliminary study the function of differential antigens in vitroIn order to preliminary study the function of different antigens,the transcriptional differences,endogenous expression and localization of antigen genes at different stages,and whether they are involved in sporozoite invasion were tested in the study.RT-PCR showed that the eight genes were transcribed at the stage of non-sporulated oocysts,sporozoated oocysts,sporozoites and merozoites,and were significantly different in different genes.The transcription levels of EtCCT2,EtHSP60,EtRACK,EtProfilin,EtSERPIN1,EtG-3-P and EtMIC1 are significantly higher expressed in the merozoite than in other stages.EtPyruvate kinase has higher transcription levels in sporulated oocysts,sporozoites and merozoites.The transcription level of EtEtMIC1 was significantly higher in sporozoite and merozoite stages than non-sporulated oocysts,sporozoated oocysts.In order to further study the functions of proteins,the mouse polyclonal antibody were prepared.The sporozoites and merozoites were immunostained by indirect immunofluorescence analysis?IFA?,using the polyclonal antibody as the primary antibody.The results showed that eight antigens were expressed in sporozoites and merozoites,and the sporozoites and merozoites could be immunostained by anti-EtMIC1,EtHSP60,EtSERPIN1 and EtCCT2 polyclonal antibodies,which suggest that they may be the membrane antigen.The ability to inhibit the sporozoites invasion of DF1 was tested In vitro.Results showed that the inhibition rate of anti-EtMIC1?38.5%?and anti-EtSERPIN1?36.7%?polyclonal antibodies were significantly higher than negative control,while it was no significant difference between other groups and control.4.Identification of E.tenella specific antigensIn order to further identify the species-specific antigens of E.tenella,the protective effects of E.tenella and E.maxima antigens were evaluated in our study.EmMIC1,EmHSP60,EmSERPIN1,Emprofilin,EmCCT2,Empyruvate kinase,EmRACK and EmG-3-P genes were cloned by PCR and the recombinant proteins were obtained.The protective efficacies of recombinant E.tenella and E.maxima proteins against E.tenella were evaluated.The results showed that the recombinant EtCCT2,Etprofilin,EtMIC1,EtPyruvate kinase and EmPyruvate kinase could significantly reduce the cecum lesion scores and oocyst shedding,significantly increase weight gain,and provide good protective efficacies?ACI:180.77,171.44,173.69,171.44 and 167.87?.EtG-3-P,EmG-3-P and EtSERPIN1 could provide limited immune protection?ACI:150.94/149.55/152.56?.In summary,EtCCT2,Etprofilin,EtMIC1,and Etpyruvate kinase can provide good immune protection against E.tenella chellenge,and only the Empyruvate kinase has strong cross-immunogenicity.These date suggested that EtCCT2,Etprofilin,and EtMIC1 may be E.tenella species-specific antigens,and pyruvate kinase may be a common antigen of E.tenella and E.maxima.5.The functions study of E.tenella species-specific antigensThe results indicate that EtCCT2 is a specific antigen of E.tenella,which may be functionally different from EmCCT2.In the present study,the function of EtCCT2 and EmCCT2 were investigated.In our previous studies,EtCCT2 can be localized on the surface of the parasite and not involved the invasion process.Therefore,EtCCT2 may perform its function through interaction with the host.In order to verify the conjecture,this study explored the interactions between EtCCT2/EmCCT2 and the host.The confocal observations found that EtCCT2 were co-localized with endogenous and exogenous chicken CCT4?GgCCT4?;co-immunoprecipitation?co-IP?experiments showed that EtCCT2 and endogenous chicken or exogenous GgCCT4 could co-precipitate.These results showed that EtCCT2 was interaction with GgCCT4.Chicken CCT is an protein complex,which contained8 subunit,and the adjacent to CCT2 are CCT4 and CCT5.Therefore,whether EtCCT2 could interacted with GgCCT5.The recombinant GgCCT5 eukaryotic expression plasmid were constructed and expressed successfully in DF1 cells.Co-localization assay showed that EtCCT2 was interacted with GgCCT5.To further investigate the effects of interaction complexes on cells,the location of the interaction complexes were examined.IFA showed that the interaction complexes aggregated in the form of granular aggregates,and entered the nucleus,eventually leading to nuclear lysis and apoptosis increased.EmCCT2 functional analysis showed that EmCCT2 only interacts with GgCCT5 and does not induce apoptosis.The difference in CCT2 function may the results in E.tenella and E.maxima resistance of different parasitic environment.In addition,our studies have found that E.tenella infection and CCT2 expression could regulate the expression level of host CCT4.Therefore,EtCCT2may be a potential virulence factor for E.tenella,and may regulate the process of host host cells through interaction with GgCCT.6.Identification of the epitopes of EtMIC1 and the key amino acid of eoitopesThe epitopes of EtMIC1 were identified by segmented expression,using anti-EtMIC1monoclonal antibodies?1-A1 and 1-H2?as primary antibodies,and the species-specific and immunogenic of epitopes were identified.Two epitopes(I:⁹¹LITFATRSK⁹99 and CTR:⁶⁹⁸ESLISAGE⁷⁰⁵)of EtMIC1 were identified successfully.Site-directed mutation analysis suggested that all epitope amino acids were essential for the reaction.Sequence alignment showed that epitope I were different among seven chicken Eimeria spp..Western blot showed that there were no cross-reactivity between the anti-EtMIC1 monoclonal antibodies and other six Eimeria spp..In order to identify the immonogenicity of the epitopes,the protective efficacies of recombinant epitope peptides were evaluated.The results showed that the recombinant I epitope peptide could significantly reduce the cecum lesions and oocyst shedding caused by E.tenella challenge,significantly increased the weight gains,and could provide the same protective efficacies as recombinant EtMIC1.In addition,the recombinant epitope I peptide could induce high levels of serum antibodies,significantly increase the level of lymphocyte transformation and proportion of CD4+lymphocyte cells.These results suggested that epitope I has good immunogenicity,and could induce humoral and cellular immunity response,and may be a species-specific epitope of E.tenella.Summary,four species-specific antigens of E.tenella and a common the antigens of E.tenella and E.maxima were screened by comparative immunoproteomics.The functional differences of the CCT2 protein in E.tenella and E.maxima were studied,which may be a potential virulence factor for E.tenella.Identification of a high immunogenic species-specific epitope of EtMIC1.Our study will provide new targets for the design and development of a new subunit vaccine of E.tenella.