Isolation And Identification Of Eimeria Tenella Exosome And Preliminary Study On The Function Of Ubiquitin

Eimeria tenella(Et)is a single-cell protozoan that specifically infects the chicken cecum.It is one of the main pathogens of coccidiosis in chickens.At present,chemical drugs are mainly used to prevention and treatment of chicken coccidiosis,but drug resistance has greatly reduced the effectiveness of the drugs.So,it is urgent to understand the parasite-host interaction mechanism and develop new prevention and cure pathways.Exosomes are membrane-like vesicles that carry a variety of bioactive substances,such as proteins,RNA and lipids.Various functions of exosomes have been reported including immunomodulation,cell migration,and cell-to-cell communication.Till now,a variety of parasites,exosomes have been identified to play an important role in the parasite-host interaction,however the exosomes of Eimeria still remains unknown.In this study,the exosome of Et(EtExo)was isolated and identified for the first time,and one of the Et Exo protein,ubiquitin(Et ubiquitin,Et Ub)(gene number: ETH00031625)was selected to furtter studily.The results provide a basis for further study of the interaction mechanism between Eimeria tenella and host.1.Isolation and identification of EtExoThe culture medium was collected after the sporozoites were cultured for 24 h.Exosomes of the culture medium were isolated by differential centrifugation.Then,EtExo was identified by transmission electron microscopy,nanoparticle tracking analysis,and Western blot.The results displayed that EtExo have a typical cup shaped vesicle structure with a diameter of about 106~108 nm was observed,which could be recognized by rabbit anti-sporozoite whole protein serum and rabbit anti-recombinant ?-elongation factor 1 serum respectively.Later,we found that EtExo could be fused with HD11 cells,while HD11 cells proliferation were significantly inhibited when the concentration at 8 ?g/mL,16 ?g/mL,and 32 ?g/mL.Then shotgun LC-MS/MS assay was performed for EtExo.After comparing with the Eimeria tenella Uniprot protein database,a total of 359 proteins were identified,including 29 exosomes commonly identified protein molecules such as Actin,Enolase,Lactatedehy drogenase,Pyruvate kinase,Elongation factor 1-alpha,Heat shock protein 90,ubiquitin;18 apicomplexan protozoan antigen molecules such as Microneme protein,Apical membrane antigen-1,Rhoptry neck protein 2,Surface Antigen and TA4;18 enzyme molecules such as Enolase,LDH and PK.GO analysis showed that EtExo proteins were mainly involved in molecular functions such as catalytic activity,binding,structural molecule activity,and transporter activity.2.Cloning and expression of the Et Ub geneIn the present study,the EtUb sequence was cloned by PCR with the cDNA of the E.tenella sporulated oocyst as template.Bioinformatics analysis showed that the EtUb gene was obtained with 777 bp sequence,encoding 258 amino acids with predicted protein molecular weight of 28.4 kDa and isoelectric point of 4.78.The deduced amino sequence had no predicted signal peptide or transmembrane region.Compared with other chicken Eimeria ubiquitin gene the homology was 70.35%-97.67%,indicated that the protein has a well conserved in chicken Eimeria.The target segments was subcloned to pGEM-28 a vector the prokaryotic recombinant expression plasmid pGEM-28a(+)-EtUb was constructed.The expression analysis in vitro showed that the protein was mainly expressed in the supernatant and the rEtUb protein was collected by His affinity chromatography column.qPCR results showed that the mRNA level of EtUb gene in merozoites and sporulated oocysts was significantly higher than that in unspored oocysts and sporozoites.3.Preliminary analysis of the function of EtUb proteinIn the study,the prepared rabbit anti-rEtUb polyclonal antibody specifically recognizes sporozoite protein,indicated that the obtained antibody had a well reactogenicity.Indirect immunofluorescence was applied to analyze the distribution of EtUb in sporozoites and second generation merozoites.Antibody invasion inhibition experiment was applied to analyze the effect of rabbit anti-rEtUb-IgG on the sporozoite BHK cells invasion.Immunoprecipitation was applied to screen the potential parasite proteins interacting with EtUb.The results revealed that the protein levels of EtUb was significantly higher expressed in second generation merozoites than the unsporulated oocysts,sporulated oocysts and sporozoites.The EtUb protein was mainly located in the top surfaces of sporozoites and second generation merozoites as shown in indirect immunofluorescence.When the anti-rEtUb-IgG concentration was 300 ?g/mL,the sporozoites invasion was significantly inhibited and the inhibition rate reached 36%.A total of 10 proteins potentially interacted with EtUb,including Myosin A and Microneme protein 3,was screened.These results were laying the foundation for further study the functions of EtUb in Et.