Expression And Localization Analysis Of Eimeria Tenella Microneme Protein 7 At Merozoite Developmental Stages

Eimeria tenella is an obligate intracellular protozoan parasites,which can be infectious to chickens of different breeds and ages.It can cause a very high fatality rate and serious threat to the development of the poultry breeding industry.A large number of studies have shown that the adhesion-related domains contained in microneme proteins can participate in extracellular functions,such as adhesion and ligand-receptor interactions.Therefore,identifying novel microneme antigens and investigating their functions are of great significance for the prevention and treatment of coccidiosis.In this study,the EtMIC7 gene of E.tenella was analyzed by bioinformatics software.The designed a pair of specific primers were used to amplify the EtMIC7157-681 bp gene containing the EGF domain from the c DNA of sporulated oocysts obtained by reverse transcription.The prokaryotic expression vector p ET30a-EtMIC7157-681 bp was constructed.The recombinant plasmid was used to obtain the fusion protein by purification after induced expression.The purified EtMIC753-227 aa protein was immunized to BABL/c mice to generate polyclonal antibodies.A positive serum was obtained by oral infection of sporulated oocysts of E.tenella.The merozoites of E.tenella were collected and purified.Immunofluorescence was performed with the merozoites immobilized on the attached glass slide,and the total protein of the merozoites was extracted for immunoblotting.The above process used the generated mouse anti-EtMIC753-277 aa polyclonal antibody as the primary antibody to detect the localization and expression of the EtMIC7 protein.The results showed that the EtMIC7 gene encodes 344 amino acids.The molecular weight of EtMIC7 protein is 36.573 k Da,and it is a hydrophilic protein.The EtMIC7 protein contains 1 transmembrane region(from aa284 to aa307 peptide)and 15 epitopes.Therefore,it is speculated that the EtMIC7 protein of E.tenella has better immunogenicity.Fusion protein obtained by purification after induced expression mostly exists in the form of inclusion bodies and has good immunoreactivity.The prepared antibody has good reactivity.The EtMIC7 protein can be expressed in the merozoites of E.tenella.The protein mainly exists in the cytoplasm and is also distributed in the anterior end of the parasite.Therefore,this study will identify whether EtMIC7 can be used as a ligand for chicken intestinal epidermal growth factor(CER)in the future.According to the characteristics of the ligand-receptor combination,it is possible to further explore the mechanism of activating multiple signal pathways in order to ensure the growth and development of parasites in host cells.At the same time,it also provides ideas for the discovery of candidate antigens for new vaccines.