Screening The Interacting Proteins Of EtCDPK3 And Primary Study On The Function Of Two Proteins

Avian coccidiosis is a highly harmful global parasitic disease caused by several Eimeria parasites in the intestines of chickens.Eimeria tenella(E.tenella)is a major causative agent of avian coccidiosis.Previous studies in our laboratory have found that E.tenella calcium-dependent protein kinases 3(EtCDPK3)is related to the invasion of host cells by the sporozoite.In this study,we used Co-Immunoprecipitation and mass spectrometry to screen for proteins that might interact with EtCDPK3.The interaction between EtCDPK3 and the E.tenella nucleoside diphosphate kinase(EtNDK)and the conserved protein of E.tenella(EtCHP)that had been screened in our laboratory was verified.In addition,the characteristics of the putative E.tenella AN1-like zinc finger protein(EtZN1-ZnFP)and EtCHP were analyzed.The effect of EtCHP and EtZN1-ZnFP was analyzed by immunoprotection assay.1.Co-IP combined mass spectrometry for screening the proteins that putative interacting with EtCDPK3The purified sporozoites were collected and the total proteins of sporozoites was extracted.Co-IP were used for screen the interacting proteins with EtCDPK3.Then the mass spectrometry was performed to analysis these proteins.The results showed that 145 proteins in the Mb group,167 in the Mb-N group,227 in the Pb group,and 189 in the Pb-N group were screened out.The proteins were sorted from the two sample groups but not in the negative control group.Six sporozoite-related proteins were obtained,including ATP synthase gama chain,phosphoglucomutase/parafusin related protein 1,nucleoside diphosphate kinase,6-phosphogluconate dehydrogenase and two conserved proteins.2.Construction of recombinant plasmids and verification of interacting proteinThe prokaryotic expression recombinant plasmids pGEX-4T-1-EtCHP and pET-28a-EtNDK and eukaryotic recombinant plasmids pcDNA3.1(+)-flag-EtCHP and pBiFC-VC155-EtNDK were constructed.The interaction between EtNDK and EtCDPK3 was verified by His Pull-down and BiFC.In addition,the interaction between EtCHP and EtCDPK3 was verified by GST Pull-down and Co-IP.All these results showed that there is no interaction between EtCDPK3 and two proteins(EtNDK and EtCHP),respectively.3.Preliminary analysis of the function of EtCHP and Et ZN1-ZnFPThe ORF sequences of the EtCHP and EtZN1-ZnFP gene were obtained by PCR amplification,respectively.Quantitative PCR showed that mRNA transcripts of EtCHP were most abundant in the sporozoite stage and the mRNA transcripts of EtAN1-ZnFP were most abundant in unsporulated oocysts and sporozoite stages.Anti-r EtCHP polyclonal antibody serum was prepared by immunizing rabbits and mice with purified rEtCHP protein,respectively.Anti-rEtAN1-ZnFP polyclonal antibody serum has been prepared in our lab.Western blot analysis showed that both of them had good reactogenicity.Indirect immunofluorescence results showed that EtCHP was mainly distributed on the surface of sporozoites and the second generation merozoites.EtAN1-ZnFP was evenly distributed in the cytoplasm of sporozoites and immature schizonts,and the expression levels of EtAN1-ZnFP decreased in the first-generation of mature schizonts.The results of in vitro invasion inhibition showed that the infection inhibition rate of anti-rEtCHP and anti-rEtAN1-ZnFP were 28% and 30%,respectively.4.Protective efficacy of rEtCHP and rEtZN1-ZnFP on E.tenella challengeImmunization and challenge infection experiments were performed to evaluate the efficacy of the purified rEtCHP and rEtAN1-ZnFP protein against E.tenella.The results showed that the mean lesion score and fecal oocyst output immunized with the recombinant protein rEtCHP and rEtZN1-ZnFP were significantly lower compared with the non-immunized chickens(P<0.05).The cytokine levels of immunized chickens with rEtCHP and rEtAN1-ZnFP protein were evaluated by quantitative ELISA.The results showed that the level of IgG in the serum of immunized chickens was significantly higher than the non-immunized control group(P<0.05).Compared with the non-immunized control group,the cytokine levels in the serum were not significantly changed except for ssCD8 after 7 days of second immunization.However,after 9 days of challenge,the ssCD4 level of rEtZN1ZnFP/ISA71(50 ug/bird)immunized group was significantly higher than the non-immunized control group(P<0.05).The levels of ssCD8 in both groups of immunized chickens with rEtZN1-ZnFP/ISA71 were significantly increased(P<0.05).The levels of IL-17 and TGF-β1 in the two groups of immunized chickens with rEtCHP/ISA71 were significantly higher than the non-immunized control group(P<0.05).These results indicated that rEtCHP and rEtZN1-ZnFP play important role in resisting E.tenella infection.