Screening And Characteristics Of Differentially Expressed Proteins Of Chicken Embryo Fibroblasts Cells Infected With Eimeria Tenella

Eimeria tenella(E.tenella)is one of the seven species of Eimeria,which is highly pathogenic.E.tenella is an obligate intracellular parasite with a complex life history and must rely on host cells to complete its life cycle.Blocking zoite invasion of cells may be an effective method to prevent and control coccidiosis.In this study,isobaric tags for relative and absolute quantitation(iTRAQ)coupled with LC-MS/MS was employed to screen differentially expressed proteins(DEPs)in chicken embryo fibroblasts(CEF)cells infected with E.tenella sporozoites for 72 h.And three differentially expressed proteins,including fatty acid binding protein 4(FABP4),glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and protocadherin 10(PCDH10),were selected to study their role in E.tenella invasion.The results provide a theoretical basis for elucidating the mechanism by which coccidia interact with the host.1.Analysis of differentially expressed proteins in CEF cells infected with E.tenella by iTRAQ technologyEimeria tenella is an obligate intracellular parasite that invades cecal epithelial cells of chickens.When infected with E.tenella,the host cells undergo corresponding changes to cope with the infection.However,research on the mechanism of host cells responds to coccidia infection is extremely limited.In this study,iTRAQ coupled with LC-MS/MS was used to screen the DEPs in CEF cells infected with E.tenella sporozoites for 72 h.A total of 3967 non-redundant distinct proteins were identified and 259 of these were the DEPs,145 up-regulated proteins and 114 down-regulated proteins were included.Gene Ontology enrichment indicated that these DEPs were mainly participate in binding activity,catalytic activity and transporter activity.KEGG pathway analysis showed that the DEPs were participated in the extracellular matrix receptor interaction,glycolysis/gluconeogenesis,focal adhesion,amino acid biosynthesis,cysteine and methionine metabolism.q-PCR were used to verify the reliability of iTRAQ proteomic data.q-PCR results showed that seven genes' mRNA transcriptional levels were consistent with iTRAQ results,but one gene were inconsistent.The findings provide a new perspective for exploring the mechanisms by which parasites interact with hosts.2.Effects of FABP4 on E.tenella sporozoites invasion of cellsA 458-bp Gallus gallus FABP4 gene was cloned and subcloned to pET-28c(+)vector to construct the prokaryotic recombinant expression plasmid pET-28c(+)–FABP4.The 18.5 kDa recombinant FABP4 protein(rFABP4)was expressed and identified by Western blotting.The expression of FABP4 in E.tenella sporozoites-infected cells were down-regulated detected by Western blotting and immunohistochemistry.While the q-PCR results showed that the expression level of FABP4 was increased in E.tenella sporozoites-infected cells.We hypothesize that the mRNA level is inconsistent with the protein level is due to the post-transcriptional modification or protein degradation.The antibody inhibition assay showed that antibodies against FABP4 at 50,100,200,300 and 400 ?g/mL had no significant effect on sporozoite invasion.BMS-309403 and transforming growth factor-?3(TGF-?3)was used to inhibit and improve expression of FABP4 in DF-1 cells,respectively,and their effect on the sporozoites invasion of cells was detected.Invasion rate in the BMS-309403-treated group was not significantly affected;however,the invasion rate in the TGF-?3-treated group declined significantly.The results show that host FABP4 plays a negative role in Eimeria invasion.3.Effects of GAPDH on E.tenella sporozoites invasion of cellsA 1123-bp Gallus gallus GAPDH gene was cloned and subcloned to pGEX-4T-1 vector to construct the prokaryotic recombinant expression plasmid pGEX-4T-1-GAPDH.The 61.7 kDa recombinant GAPDH protein(rGAPDH)was expressed and identified by Western blotting.The expression of GAPDH in E.tenella sporozoites-infected cells were up-regulated detected by q-PCR and immunohistochemistry.While Western blotting showed that the expression of GAPDH has no significant difference between E.tenella sporozoites-infected and non-inected cells.The antibody inhibition assay showed that antibodies against GAPDH at 50,100,200,300 and 400 ?g/mL can significantly inhibited sporozoite invasion compared with the same does of normal rabbit IgG.The results show that host GAPDH plays an active role in Eimeria invasion.4.Effects of PCDH10 on E.tenella sporozoites invasion of cellsA 1631-bp Gallus gallus PCDH10 gene was cloned and subcloned to pCold ? vector to construct the prokaryotic recombinant expression plasmid pCold ?-PCDH10.The 63.2 kDa recombinant PCDH10 protein(rPCDH10)was expressed and identified by Western blotting.The expression of PCDH10 in E.tenella sporozoites-infected cells were down-regulated detected by Western blotting and immunohistochemistry.The antibody inhibition assay showed that antibodies against PCDH10 at 50,100,200,300 and 400 ?g/mL had no significant effect on sporozoite invasion.More researches are needed to study the role of PCDH10 in the process of E.tenella invasion.