【摘要】 旨在通过转录组学技术筛选出猪圆环病毒感染宿主细胞的差异表达基因(DEGs),为揭示猪圆环病毒2型(PCV2)引起猪宿主免疫细胞产生免疫抑制的分子机制提供理论依据。以断奶仔猪脾脏为试验材料，制成猪脾淋巴细胞悬液，PCV2体外感染细胞，通过转录组学测序技术筛选DEGs,进行基因本体论(GO)、京都基因与基因组百科全书(KEGG)功能分析，对关键通路采用基因集富集分析(GSEA)和进行通路标色，挑选差异表达的基因，通过RT-qPCR验证转录组测序结果的准确性，使用Western blot技术检测重要基因的蛋白表达水平。转录组测序结果显示，PCV2感染后共捕获到15 926个差异基因，其中305个差异基因表达显著上调，163个差异基因表达显著下调；GO和KEGG富集结果显示，差异基因与免疫应答等相关，主要富集在细胞因子-细胞因子受体相互作用、IL-17信号通路、风湿性关节炎、TNF信号通路、趋化因子信号通路、JAK-STAT、Toll样受体信号通路等；GSEA富集分析发现，IL-17信号通路富集于78个基因，主要包括AMCF-II、IL17F、IL13、CXCL10、CCL17等34个显著不同的基因，整体通路发生上调，TNF信号通路富集于95个基因，主要包括LTA、CXCL10、MMP9、CXCL2、CCL2等40个显著不同的基因，整体通路发生上调；RT-qPCR结果与测序结果一致，表明测序可靠；Western blot检测结果显示，PCV2感染后IL-17、TNF-α水平显著升高(P<0.05),CXCL2、IL-6水平也有所升高。转录组测序结果及后续试验结果表明，PCV2能通过上调宿主细胞IL-17、TNF等信号通路基因和蛋白表达水平影响下游炎症通路，进而引起机体产生免疫抑制。更多还原

【Abstract】 This study aims to screen out the differentially expressed genes(DEGs) in host cells infected with porcine circovirus(PCV) using transcriptome sequencing technology, providing a theoretical basis for elucidating the molecular mechanisms underlying the immunosuppression induced by porcine circovirus type 2(PCV2) in pig host immune cells.The spleen of weaned piglets was used as the experimental material to prepare pig spleen lymphocyte suspension, and PCV2 was used to infect the cells in vitro.DEGs were screened by transcriptome sequencing technology, and gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) functional analysis were performed on differentially expressed genes.Key pathways were analyzed by gene set enrichment analysis(GSEA) and pathway colors were performed.The accurate characterization of transcriptome sequencing results was verified by RT-qPCR,and the protein expression levels of important genes were detected by Western blot.Transcriptome sequencing results showed that a total of 15 926 differentially expressed genes were captured after PCV2 infection, of which 305 differentially expressed genes were significantly up-regulated and 163 differentially expressed genes were significantly down-regulated.GO and KEGG enrichment results showed that differential genes were related to immune response, mainly enriched in cytokine-cytokine receptor interaction, IL-17 signaling pathway, rheumatoid arthritis, TNF signaling pathway, chemokine signaling pathway, JAK-STAT,Toll-like receptor signaling pathway, etc.The results of RT-qPCR were consistent with the sequencing results, indicating that the sequencing was reliable.The results of Western blot showed that the levels of IL-17 and TNF-α were significantly increased after PCV2 infection(P<0.05),and the levels of CXCL2 and IL-6 were also increased.Through transcriptome sequencing, it was found that PCV2 can affect downstream pathways through IL-17 and TNF signaling pathways, and then cause immunosuppression in the body.更多还原