Study On The Role Of E.Maxima Rhomboid-like Proteins In Coccidia Invasion

Chicken Coccidiosis is an important parasitic disease caused by Eimeria coccidia,resulting in chickens weight gain rate and egg production rate decreased.Coccidiosis caused huge economic loss to poultry industry worldwide and the annual economic losse is estimated to exceed 3 billion US dollars.The traditional control method for chicken coccidiosis is mainly dependent on the use of anti-coccidiosis drugs and live vaccines.The increasing relevant regulations and decrees on the use of anti-coccidiosis drugs and the high cost of live coccidiosis vaccines have prompted people to seek new ways to treat coccidiosis.In the preliminary study of this experiment,the cDNA library of Eimeria maxima was constructed,and then the immunoprotective gene E.maxima Rhomboid-like Proteins(EmRP)was selected by immunizing the library.According to reports,the Rhomboid-like Proteins exists in almost all creatures,and plays a variety of biological functions.Especially in Apicomplexan parasites,it is mainly related to the invasion of parasites by cuting the adhesion factor of the parasites.In this study,the roles of E.maxima Rhomboid-like Proteins on coccidiosis were studied in the following aspects:1 Clone expression of EmROMs gene and sporozoite localizationIn order to obtain all the Rhomboid-like Proteins of Eimeria maxima,four pairs of primers were designed according to the hypothetical Rhomboid-like Proteins in GenBank.Four genes were amplified by PCR using the cDNA of Eimeria maxima,and then subjected to sequence analysis and prokaryotic expression.The anti-rat polyclonal antibody was prepared.Finally,sporozoite localization was analyzed by immunofluorescence.The complete sequence of four genes was obtained.We named them EmROM1,EmROM2,EmROM3 and EmROM5,respectively,according to their similarity to Rhomboid-like Proteins of the Toxoplasma gondii.The transmembrane analysis showed that they all contained conserved regions of Rhomboid-like Proteins.Sporozoite localization demonstrated that all four genes express in the sporozoites and distribute on the surface of sporozoites.2 Screening of the Interaction Gene between EmROMs and EmMICsIn order to clarify the interaction gene between EmROMs and EmMICs,the authors used DUALhunter starter yeast two-hybrid system,pDHB1-EmROMs as bait vector,pPR3-N-MICs,pPR3-N-AMA1 as prey vector.Four rounds of screening experiments were conducted.The results show that EmROM1 and EmROM3 interact with EmMIC1,and EmROM1,EmROM2,EmROM3 and EmROM5 interact with EmMIC2.3 Study on the cleavage of EmMICland EmMIC2 by EmROMsIn order to verify cleavage between EmROMs and EmMIC1,EmMIC2,the eukaryotic expression plasmids pVAX1-Flag-EmROMs and p VAX1-Flag-EmMIC1,pVAX1-Flag-EmMIC2 were constructed and transfected into Hela.The expression and cleavage of two proteins was detected by immunofluorescence and western blot.The results showed that EmROM1 and EmROM3 could cleave EmMICl.4 Study on immunogenicity of EmROM3In order to explore the immunogenicity of EmROM3,we constructed the eukaryotic expression plasmid pVAXl-EmROM3,and the first immunization was immunized with 2 weeks old chicks.The second immunization were immunized at 3 weeks of age,The serum IgG levels were detected by ELISA,and the levels of CD4+ and CD8+ of T lymphocytes were detected by flow cytometry.The results show that pVAX1-EmROM3 can stimulate the increase of cytokines and proliferation of T lymphocyte subtypes,which can be used as a candidate gene for Eimeria maxima DNA vaccine.