摘要：

为研究效应蛋白Hcp2b在禽致病性大肠杆菌(APEC)感染雏鸡过程中对脾脏的转录组学影响,利用兽医病理生物学与疫病防控安徽省重点实验室构建保存的hcp2b基因缺失株Δhcp2b及回复株Chcp2b肌肉注射7日龄雏鸡(以AE17菌株为阳性对照),感染24 h后采集精神沉郁的雏鸡脾脏检测组织载菌量并制作病理切片。选取缺失株Δhcp2b及野生株AE17感染脾脏组织进行转录组学测序,采用Real-time PCR进行测序结果鉴定;而后对差异表达基因进行GO分析、KEGG通路富集分析。结果显示,野生株AE17、缺失株Δhcp2b及回复株Chcp2b在脾脏组织载菌量差异不明显。组织切片观察发现,野生株AE17和缺失株Δhcp2b脾脏均出现组织间隙扩大,有充血现象出现。转录组学分析发现,缺失株Δhcp2b感染雏鸡脾脏后,诱导了脾脏基因mRNA的差异表达,筛选到515个差异表达基因(330个基因下调表达,185个基因上调表达)。Real-time PCR结果发现,Δhcp2b感染雏鸡后,脾脏中IL22、TNFRSF6B、TNFRSF8基因mRNA表达量下调,与测序结果变化趋势一致。GO分析发现,感染24 h雏鸡脾脏差异基因富集在膜、膜的组成、细胞外间隙部分等条目。KEGG分析发现,脾脏差异基因显著富集在内质网蛋白质加工过程的信号通路中。hcp2b基因对APEC在定植脾脏无影响,hcp2b通过影响机体免疫器官中脾脏的内质网蛋白质加工过程的信号通路来参与致病过程。

关键词: 雏鸡, 禽致病性大肠杆菌, Hcp, 脾脏, 内质网蛋白质加工通路

Abstract:

In order to study the effect of the effector protein Hcp2b on the transcriptomics of the spleen during the infection of Avian pathogenic E.coli(APEC)in chicks.The hcp2b-deletion strain Δhcp2b and the reverted strain Chcp2b(with AE17 strain as a positive control)constructed and preserved in the Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control were used to inject 7-day-old chicks intramuscularly.After 24 h,the spleen of depressed chicks was collected to detect the tissue load and make pathological sections.The deletion strain Δhcp2b and the wild strain AE17 were selected to infect spleen tissue for transcriptomics sequencing.Also,Real-time PCR was used to identify the sequencing results.Moreover,GO analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes.The results illustrated that the wild strain AE17,the deleted strain Δhcp2b and the reverted strain Chcp2b had no significant difference in the tissue load of the spleen.Observation of tissue sections that the spleens of wild strain AE17 and deletion strain Δhcp2b both suggested enlarged interstitial spaces and hyperemia.Transcriptomics analysis revealed that the deletion strain Δhcp2b induced the differential expression of spleen gene mRNA in chicks.515 differentially expressed genes were screened(185 genes were up-regulated and 330 genes were down-regulated).Real-time PCR confirmed that the expression of IL22,TNFRSF6B,and TNFRSF8 genes in the spleen was down-regulated after the deletion strain Δhcp2b infected chicks,which was consistent with the trend of the sequencing results.At 24 h,GO analysis found that the chick spleen differential genes were significantly enriched in items such as membrane,membrane composition,and extracellular space.KEGG analysis found that differential genes in the spleen were significantly enriched in the pathway of endoplasmic reticulum protein processing.The hcp2b gene had no effect on APEC colonization of spleen,the hcp2b participates in the pathogenic process through the signal pathway that affects the endoplasmic reticulum protein processing of the spleen in the body's immune organs.

Key words: Chicken, Avian pathogenic E.coli, Hcp, Spleen, Endoplasmic reticulum protein processing pathway