羊源D型多杀性巴氏杆菌感染小鼠脾脏转录组分析

Transcriptome Analysis of Spleen of Mouse Infected with Pasteurella multocida Type D from Goat

试验旨在探索羊源D型多杀性巴氏杆菌入侵宿主脾脏组织中引发的免疫应答途径。首先利用羊源D型多杀性巴氏杆菌(HN01菌株)感染小鼠,建立羊源D型多杀性巴氏杆菌感染的动物模型;之后利用转录组测序技术获得感染小鼠与正常小鼠的脾脏转录组数据,并使用COG、KOG、eggNOG、GO、KEGG数据库对测序结果中的差异表达基因(differentially expressed genes,DEGs)进行功能注释与分析,同时对于显著富集到关键免疫通路的差异表达基因使用STRING软件和KEGG mapper进行蛋白互作分析,筛选出核心通路中起关键作用的基因;最后选取关键的10个基因进行实时荧光定量RT-PCR验证。转录组分析结果显示,与正常组相比,感染组中筛选出3380个差异表达基因(P<0.01,log_(2)|FoldChange|≥0.5),其中1691个基因上调,1689个下调。基因功能富集分析结果表明,感染组脾脏中的差异表达基因主要发挥信号转导的功能,其主要参与的生物途径包括细胞因子与细胞因子受体互作通路、趋化因子信号通路、HIF-1信号通路、TNF信号通路。蛋白互作分析筛选出约28个核心差异表达基因,结合实时荧光定量RT-PCR验证后,其中9个基因的表达结果与测序一致,分别是C3、Cd4、Cxcl13、Lck、Gnai1、Grap2、IL-6、Cxcr6及Serping1基因。本研究初步证明了脾脏在抵抗多杀性巴氏杆菌入侵中参与了一系列免疫应答反应,为进一步研究羊源D型多杀性巴氏杆菌与宿主之间相互作用的分子机制奠定理论基础。

In order to illustrate the transcriptomic profile of spleen in mice upon Pasteurella multocida infection,the mouse model of P.multocida infection was established by intraperitoneal injection.Transcriptome sequencing technology was performed to obtain spleen transcriptome data of infected and normal mice.Furthermore,COG,KOG,eggNOG,GO,KEGG database were utilized to accomplish functional annotation and the differentially expressed genes(DEGs)analysis.In order to screen out the genes that played the key role in immune pathways,DEGs which were significantly enriched in several immune pathways,were used to perform protein interaction analysis with STRING software and KEGG mapper.Finally,10 key genes were randomly selected for Real-time RT-PCR verification.Transcriptome analysis results showed that compared with the normal group,3380 DEGs were screened in the infection group(P<0.01,log_(2)|FoldChange|≥0.5),of which 1691 DEGs were up-regulated and 1689 DEGs were down-regulated.Gene function enrichment analysis showed that DEGs in spleen of the infected group mainly played the function of signal transduction.The cytokine-cytokine receptor interaction pathway,chemokine signal pathway,HIF-1 signal pathway and TNF signal pathway were significantly enriched in the context of P.multocida infection.Protein interaction analysis screened out 28 core DEGs.After verification with Real-time RT-PCR,the expression of 9 genes were consistent with sequencing results,namely C3,Cd4,Cxcl13,Lck,Gnai1,Grap2,IL-6,Cxcr6 and Serping1 genes.This study demonstrated that the spleen played the essential immune role in resisting the invasion of P.multocida and could provide valuable information on the molecular mechanism of the interaction between goat-derived P.multocida and the host.