Identification Of The Interaction Between Eimeria Tenella Apical Membrane Antigen 1 And Rhoptry Neck Protein 2 And Virtual Screening Of Its Blockers

Chicken coccidiosis is an intracellular protozoal disease caused by Eimeria spp.,which mainly damages the intestines of chickens.Chicken coccidiosis is a globally distributed parasitic disease that causes huge economic losses to the chicken industry.At present,the main prevention and control measures for coccidiosis are drug prevention,and long-term,large-scale drug inputs have caused coccidiosis resistance and drug residue problems.Although the development and application of coccidiosis oocyst vaccines is a new strategy for the prevention of coccidiosis,oocyst vaccines also have difficulties in preparation and preservation,as well as the virulent return of vaccine strains.The main reason for these problems is that the mechanism of chicken coccidia invasion into host cells is not yet clear.Invasion of intestinal epithelial cells by sporozoites is a prerequisite for coccidiosis infection,so blocking sporozoite invasion early is a viable option for the prevention of coccidiosis.Several studies have shown that the invasion of Apicomplexa is a series of complex processes,which guided by the interactions between proteins such as microneme protein and rhoptry protein.In the study of invasion of Plasmodium falciparum and Toxoplasma gondii tachyzoites,the moving junction structure formed by the interaction between apical membrane antigen-1 and the rod protein RON2/4/5/8,which plays an important role in the connection between parasites and host cells.There are few studies on the mechanisms of chicken coccidia invading intestine epithelial cells,and the interaction between Et AMA1 and EtRON2 has not been confirmed.Therefore,we used yeast two-hybrid,GST-Pull down,co-immunoprecipitation,and bimolecular fluorescence complementation to identify the the interaction between EtAMA1 and EtRON2.Based on the above research,we sought for potential blockers that interaction between EtAMA1 and EtRON2 through virtual screening technology.These studies provide a reference for the development of new anticoccidial drugs and vaccine targets.Identification of the interaction between EtAMA1 and EtRON2 by yeast two-hybrid: Using the cDNA of Eimeria tenella sporozoites as template,the EtAMA1 and EtMIC2 genes were amplified by PCR.The yeast two-hybrid bait vector pGBKT7-EtAMA1 and the prey vector p GADT7-EtRON2 were constructed and transformed into yeast Y2 HGold strain to observe the growth of yeast on auxotrophic selection medium.Both EtAMA1 and EtRON2 had no auto-activation and toxicity on the yeast two-hybrid system,and pGBKT7-EtAMA1/pGADT7-EtRON2 co-transformed group could activate yeast two-hybrid reporter system.Identification of the interaction between EtAMA1 and EtRON2 by GST-Pull down assay: The GST-EtAMA1 and His-EtRON2 fusion proteins were expressed in E.coli and purified for GST-Pull down assay.The interaction between EtAMA1 and EtMIC2 has been identified by GST-Pull down.Identification of the interaction between EtAMA1 and EtRON2 by Co-IP: The eukaryotic expression vectors pcDNA3.1-His-EtAMA1 and pcDNA3.1-HA-EtRON2 had been constructed and transfected into HEK-293 T cells,and the cell lysate was collected and subjected to co-immunoprecipitation(Co-IP).EtAMA1 and EtRON2 protein can be co-expressed in HEK-293 T cells,and the interaction between EtAMA1 and EtRON2 can be identified by Co-IP.Identification of the interaction between EtAMA1 and EtRON2 by Bi FC: We constructed the bimolecular fluorescence complementation(BiFc)vectors pEtAMA1-Myc-LC151 and p EtRON2-HA-KN151 and transfected into HEK-293 T cells.The confocal laser scanning microscope was used to observe the fluorescence of transfected cells.Red fluorescence was observed in the EtAMA1 and Et EtRON2 co-transfected cells in the bimolecular fluorescence complementation.Screening for the blockers that interact with EtAMA1 and EtRON2 by virtual screening: Six candidate blockers of EtAMA1 and EtRON2 interactions were obtained through molecular docking and virtual screening.The results of in vitro experiments showed that the invasion inhibition rates of ZINC5416294,ZINC5759756 and ZINC144203594 against sporozoites of E.tenella were 44.07%,39.68% and 42.85%.The interaction between EtAMA1 and EtRON2 was identified by yeast two-hybrid,GST-Pull down,Co-IP and Bi Fc.This study provides the theoretical basis for revealing the molecular mechanism of microneme protein in the invasion of chicken coccidian into host cells.The three compounds obtained through virtual screening have certain inhibitory effects on the invasive of E.tenella sporozoites,and this study provides a reference for the development of new drug targets.