基于iTRAQ技术分析柔嫩艾美耳球虫感染后CEF细胞的差异表达蛋白

ANALYSIS OF DIFFERENTIALLY EXPRESSED PROTEINS IN CEF CELLS INFECTED WITH EIMERIA TENELLA BY ITRAQ TECHNOLOGY

柔嫩艾美耳球虫是一种严格的细胞内专性寄生虫,可主动入侵鸡的盲肠上皮细胞。当受到柔嫩艾美耳球虫感染时,宿主细胞会发生相应的变化以应对感染引起的损伤。迄今为止,对宿主细胞应对球虫感染机制的研究极其有限。本研究利用相对和绝对定量同位素标记(iTRAQ)技术结合液相色谱串联质谱(LC-MS/MS)技术分析了感染柔嫩艾美耳球虫子孢子72 h后原代鸡胚成纤维(CEF)细胞的蛋白质组表达变化,结果共鉴定出3967个非冗余蛋白质,与不感染组相比,感染组有259个蛋白质的表达量发生了明显变化,其中上调蛋白质145个和下调蛋白质114个;GO和KEGG通路分析结果发现,这些差异表达蛋白具有结合活性、催化活性、转运子活性等分子功能,主要参与细胞外基质受体互作、糖酵解/糖异生、粘着斑、氨基酸的生物合成、半胱氨酸和蛋氨酸代谢等通路;qPCR结果发现,8个基因的mRNA转录水平变化与iTRAQ结果一致,2个基因的不一致;Western blot结果发现感染组细胞的FABP4蛋白表达量显著降低,与iTRAQ结果中蛋白表达水平变化相一致。本研究结果为柔嫩艾美耳球虫与宿主之间互作关系的深入研究提供了重要的基础数据。

Eimeria tenella is an obligate intracellular parasite that actively invades cecum epithelial cells of chickens.When E.tenella infects host cells,the host develops the corresponding changes to deal with damages caused by this infection.To date,the mechanism of how the host cells responds to an E.tenella infection is limited.In this study,isobaric tags for relative and absolute quantitation(iTRAQ)coupled with LC-MS/MS was used to screen the differentially expressed proteins(DEPs)in chicken embryo fibroblast(CEF)cells at 72 h post infection with E.tenella sporozoites.In total,3967 non-redundant distinct proteins were identified and 259 of these proteins were identified as DEPs,including 145 up-regulated proteins and 114 down-regulated proteins.Gene ontology enrichment indicated that these DEPs mainly participated in binding,catalytic and transporter activities.KEGG pathway analysis showed that the DEPs were involved in the extracellular matrix receptor interaction,glycolysis/gluconeogenesis,focal adhesion,amino acid biosynthesis,cysteine and methionine metabolism.Subsequently,qPCR and Western blot were used to verify the reliability of iTRAQ proteomic data.The qPCR results showed that mRNA levels of eight transcriptional genes were consistent with iTRAQ analysis and two genes were inconsistent.Western blot showed that FABP4 protein expression level in infected cells was significantly decreased,which was consistent with iTRAQ results.Collectively,the data in this study provided an important basis to further analysis of E.tenella host-cell interactions.