Screening And Verifying The Interacting Proteins Of EtCDPK4 And Characteristic Analysis On The Function Of Four Proteins

Eimeria belongs to apicomlplexa protozoan,calcium-dependent protein kinases(CDPKs)are important effector molecules in the Ca2+ signaling pathway of the apicomlplexa protozoan,which control important physiological processes such as sliding movement of the parasite,cell invasion and reproduction of the parasite.Apart from this,CDPKs only exist in plants,green algae,ciliates.Since there are no CDPKs detected in the host,study the functions of CDPKs of Eimeria,and analysis its role in the Ca2+ signal pathway,will provide important molecules for the study of new therapeutic drugs and the development of new vaccines.Based on our previous research,we screened proteins that putative interacting with EtCDPK4,analyzed the characteristics of these potential interacting proteins and verified their interactions.1.Co-IP,His pull down with mass spectrometry to screen the interacting proteins with EtCDPK4We purified the sporulated oocysts of E.tenella and collected the soluble proteins of sporulated oocysts.By Co-IP and His pull down with mass spectrometry,four putative EtCDPK4 interacting proteins were obtained,that are Elongation factor 1-alpha(Et EF1?),Translation initiation factor e IF-5A(Ete IF-5A),Pyrroline-5-carboxylate reductase(Et PYCR),and 14-3-3 protein(Et14-3-3).2.Cloning,prokaryotic expression and bioinformatics analysis of four putative interacting proteins with EtCDPK4Using cDNA of E.tenella sporulated oocysts as a template,we amplified the ORF sequences of Et EF1?,Ete IF-5A,Et PYCR,and Et14-3-3.Bioinformatics analyse shows that the four gene separately encodes 450,161,173 and 277 amino acids with predicted molecular mass of 49.1 k Da,17.3 k Da,18.5 k Da and 31.7 k Da.The isoelectric points were 4.7,5.35,6.08 and 4.83,respectively.Sequence analysis found that all the proteins encoded by the four genes have no signal peptides and transmembrane domains,but both contain N-myristoylation sites.So we proposed these four proteins maybe interact with EtCDPK4 to inhibit invasion of E.tenella.3.Preliminary analysis of characteristics and functions of four genes of E.tenellaThe transcription and translation level were analyzed using real-time quantitative PCR and western blot.The results showed that the m RNA transcription levels of Et EF1? were higher in the sporozoite and second-generation merozoite stages than unsporulated oocysts and sporulated oocysts,and the translation level was highly expressed in unsporulated oocysts and merozoites.The highest level of Ete IF-5A m RNA transcription was in the second-generation merozoites,and the highest level of expression in the unsporulated oocyst.While the transcription and translation levels of Et PYCR were highest in the unsporulated oocysts,the m RNA transcription level of Et14-3-3 was highest in the sporozoite,and the translation level is higher in the unsporulated oocyst than in the other stages.Indirect immunolocalization found that Et EF1? and Et14-3-3 were mainly located at anterior of sporozoites and merozoites.Ete IF-5A and Et PYCR are mainly located at the apical of Spz.As the sporozoites develop in the cell,the fluorescence intensity of Et EF1? and Et PYCR gradually brightens while Et14-3-3 got darken.Ete IF-5A fluorescence intensity is brighter when sporozoites develop into trophozoites,weakened when developed into immature schizont,and after the mature schizont is developed,the intensity is increased.The results of invasion inhibition showed that rabbit anti-r Ete IF-5A,anti-rr Et PYCR and anti-r Et EF1? polyclonal antibodies can effectively inhibit sporozoite invasion of DF-1 cells,and the inhibition rates were 48%,58% and 22%,respectively,while rabbit anti-r Et14-3-3 antibodies have no inhibitory effect.4.Validation of EtCDPK4 and potential interacting proteinsThe eukaryotic recombinant plasmids pBiFC-VN155-Et EF1?,pBiFC-VN155-Ete IF-5A,pBiFC-VN155-Et PYCR and pBiFC-VN155-Et14-3-3 were constructed,and then co-transfected with pBiFC-VC155-EtCDPK4 into 293 T cells for BiFC assay,respectively.The results showed that the obvious green fluorescence can be seen under the microscope in the 293 T cells co-transfected pBiFC-VN155-Ete IF-5A and pBiFC-VN155-Et14-3-3 with pBiFC-VC155-EtCDPK4.On the contrary,the cells transfected by pBiFC-VN155-Et EF1?,pBiFC-VN155-Et PYCR with pBiFC-VC155-EtCDPK4 did not show obvious fluorescence,indicating that there was interaction between Ete IF-5A,Et14-3-3 and EtCDPK4,but not between Et EF1?,Et PYCR and EtCDPK4.For the two proteins verified positive by BiFC,Ig G extracted from rabbit anti-r EtCDPK4 serum was used as bait protein,r Ete IF-5A and r EtCDPK4,r Et14-3-3 and r EtCDPK4 were used as prey proteins,and Co-IP was used for verification.Meanwhile,r Ete IF-5A or r Et14-3-3 was used as bait protein,and r EtCDPK4 was used as prey protein,then further verified by GST pull down.The results showed that two obvious bands were seen on the PVDF membrane of the experimental group of r EtCDPK4 and r Ete IF-5A,r Et14-3-3,which is consistent with the results of BiFC.These results indicated that Ete IF-5A and Et14-3-3 have significant interactions with EtCDPK4,while the interactions of Et EF1? and Et PYCR with EtCDPK4 have not been confirmed.Co-localization of the two interacting proteins with EtCDPK4 revealed that they were located on the same position of sporozoites.The results indicated that Ete IF-5A and Et14-3-3 may play a synergistic role with EtCDPK4 in the invasion of host cells by sporozoites.These results laid the foundation for further research on the mechanism of action of EtCDPK4 with interacting proteins and the functions of these proteins in the life history of E.tenella.