Screening Of The Interaction Receptor Of EtRON2 In Host And Its Role During The Invasion Of Host Cells By Eimeria Tenella

Coccidiosis is a parasitic disease caused by Eimeria spp.in chicken intestinal epithelial cells.There are many species of Eimeria,however among them;Eimeria tenella is more common and highly pathogenic.This obligate intracellular parasite is widespread and seriously harms the intensive chicken production in the world.At present,the main prevention and control measures of coccidiosis are the use of anti-coccidial drugs and live coccidiosis oocysts vaccine.Although the application of live vaccine is a new strategy for the prevention and control of coccidiosis,but it has some disadvantages such as the difficulty in preparation,preservation and the strong virulence of vaccine strains.Therefore,there is an urgent need to develop new anti-coccidial drugs and molecular vaccines to solve the problems.The fundamental cause of these problems is the lack of understanding of the interaction mechanism between coccidia and host cells.It has been found that the rod-like protein RON2 plays an important role in the invasion of host cells by toxoplasma,cryptosporidium and schistosomes.However,the detailed interaction mechanism between coccidia and host cells has not been systematically understood,which makes the development of anti-coccidial drugs and vaccines even more difficult.Therefore,in this study,a secretory protein EtRON2 was used.Mass spectrometry technique was applied to study interaction of EtRON2 protein in the invasion of host cell receptors,and use the cells were positioning technology to verify the two interaction,on this basis,using blocking antibodies test,Real-time RT PCR technology research both interactions such as induced host cell F-actin gathered on the invasion of insect body important role,for chicken ball insect drugs and new vaccine targets research theory basis.1.Using prokaryotic expression with the protein GST label EtRON2 preparation of specific antibodies,based on the cultivation of the DF 1 ball in vitro cell model,through the GST pull-down,mass spectrometry technology for screening of host cell receptor,the result shows that the results of screening for confidence level is higher than 95%and protein peptides number greater than 2 potential interactions,a total of 263 successfully screened EtRON2 associated with the invasion of protein interactions and induction of the host cell F-actin gathered receptor cells protein 1(Annexin A2),it lays a foundation for further elucidation of the molecular mechanism that EtRON2 plays an important role in coccidia invasion of host cells.2.The interaction between the EtRON2 protein and the receptor was verified by subcellular localization.Western blot validation model was constructed with EtRON2 antibody,and DF-1 cells were infected with a flag-labeled vector.The results showed that flag-Annexin A2 transfection was successful in both uninfected and infected coccidia.When contact with DF-1 cell after 30 minutes,the subcellular localization process,found that EtRON2 green fluorescent signal with Annexin A2 red fluorescence signal overlap,shows that in the process of ball insect invasion of host cell,the host cell Annexin A2 protein aggregation in ball insect spore surface,validated both in sporozoite interactions in the process of the host cell invasion.3.Respectively using EtRON2 and Annexin A2 specific antibody pairs of spores and host cells,the qRT-PCR polymerase chain reaction was used to detect the chicken ball insect invasion of host cell.The results showed that compared with the normal sporozoite cells of normal DF-1 invasion of host cell F-actin aggregation was decreased significantly,and sporozoite rate of the invasion of the DF-1 is decreased obviously.The expression and transcription levels of Annexin A2 protein in MDBK cells are higher than those in DF-1 and HCT-8 cells;Compared with DF-1 cells,MDBK cell invasion activity was increased,and the invasion rate of HCT-8 cells was reduced.The above results indicate that EtRON2 interacts with the host cell protein Annexin A2,and the interaction between the two plays an important role in preventing the invasion of coccidia into host cells,which provides a theoretical foundation for the development of new anti-coccidial drugs and vaccines.