堆型艾美耳球虫棒状体蛋白17对鸡胚成纤维细胞凋亡的影响OA

To investigate the mechanism by which Eimeria acervulina rhoptry organelle protein 17(Ea ROP17)affects apoptosis in chicken embryo fibroblast(DF-1)cells,a eukaryotic expression vector Ea ROP17-pEGFP-N1 was constructed and transfected into DF-1 cells,forming the recombinant plasmid transfection(Ea ROP17-pEGFP-N1)group.The Blank control(CTRL)group and Empty vector transfection(pEGFP-N1)group were also established.Green fluorescence was observed under an inverted fluorescence microscope,and Western blot(WB)analysis was used to verify protein expression.DF-1 cells were then divided into four groups:CTRL group,Recombinant plasmid-treated(ROP17+LPS)group,Empty vector-treated(EGFP-N1+LPS)group,and Model(LPS)group.Flow cytometry was used to detect apoptosis rates in the four groups,and real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to examine the apoptosis pathway regulated by Ea ROP17.The results showed that the Ea ROP17-pEGFP-N1 eukaryotic expression vector was successfully constructed.Under the fluorescence microscope,green fluorescence was observed in both the Ea ROP17-pEGFP-N1 and pEGFP-N1 groups but not in the CTRL group.WB analysis confirmed successful transfection and expression of the recombinant Ea ROP17-pEGFP-N1 plasmid in DF-1 cells.The apoptosis model was successfully established,and flow cytometry results showed that,compared with the LPS group,the total apoptosis rate in the ROP17+LPS group was significantly reduced(P<0.05).RT-qPCR analysis revealed no significant difference in the expression of cytochrome c(Cyt c)mRNA between the ROP17+LPS and LPS groups(P>0.05),while the mRNA expression levels of Caspase-3,Caspase-8,and Caspase-9 were significantly downregulated(P<0.05,P<0.001,or P<0.000 1).These findings suggest that Ea ROP17 can inhibit host cell apoptosis,likely via the death receptor signaling pathway rather than the mitochondrial pathway.This study provides a scientific reference for the studies on the pathogenic mechanisms of avian coccidia.