持续感染口蹄疫病毒VP1蛋白对细胞转录组的影响  

Effects of VP1 Protein in Persistently Infection with Foot-andmouth Disease Virus on Cell Transcriptome

作　　者：杨黎 刘萍 韦美华 柯雄锋 刘丽清 宋静静 方莹 唐欢[1,2,3] 孔令保 辛秀[1,2,3] YANG Li;LIU Ping;WEI Meihua;KE Xiongfeng;LIU Liqing;SONG Jingjing;FANG Ying;TANG Huan;KONG Lingbao;XIN Xiu(Institute of Pathogenic Microbiology,Jiangxi Agricultural University,Nanchang 330045,China;College of Biological Science and Engineering,Jiangxi Agricultural University,Nanchang 330045,China;Nanchang Key Laboratory of Animal Virus and Genetic Engineering,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学病原微生物研究所,江西南昌330045 [2]江西农业大学生物科学与工程学院,江西南昌330045 [3]南昌市动物病毒与基因工程重点实验室,江西南昌330045

出　　处：《江西农业大学学报》2022年第2期411-421,共11页Acta Agriculturae Universitatis Jiangxiensis

基　　金：江西省自然科学基金青年项目(20192BAB214001)。

摘　　要：【目的】通过转录组测序(RNA-Seq)方法分析持续感染口蹄疫病毒结构蛋白VP1诱导的PK-15细胞基因表达谱的变化,探索VP1蛋白对宿主细胞信号通路的影响。为后续开展VP1影响FMDV持续感染相关机制研究提供基础数据和理论支持。【方法】PK-15细胞分别转染pCAGGS-HA(对照载体)、pCAGGS-HA-Pi-VP1质粒,转染36 h后,Western blot检测VP1蛋白表达,同时提取两组细胞总RNA进行转录组测序(每组3个重复,2组6个样本)。将测序所得的序列进行过滤比对,获得差异表达基因后,对差异表达基因进行了生物信息学分析。采用qRT-PCR验证关键差异表达基因的表达。【结果】RNA-Seq共鉴定出3376个差异表达基因(DEGs)|log2(Fold Change)|>0和padj<0.05,其中上调基因1744个,下调基因1632个。许多参与免疫反应和炎症的效应基因差异表达。GO和KEGG富集分析显示,差异基因GO条目绝大部分与核糖体有关,而KEGG富集中涉及了与天然免疫相关的信号通路,如TNF信号通路、Toll受体信号通路。【结论】pCAGGS-HA-Pi-VP1转染PK-15细胞之后,改变了细胞的表达谱,获得的DEGs及其功能注释信息有助于理解其对宿主细胞的影响,为后续开展VP1影响FMDV持续感染相关机制研究提供基础数据和理论支持。[Objective]Through transcriptome analysis,this study aims to analyze the gene expression profile of PK-15 cells transfected with VP1 gene of persistently infected with FMDV,and its possible signal pathways,thus providing data and theoretical foundation for future studies on the mechanisms of VP1 affecting FMDV persistent infection.[Method]PK-15 cells were transfected with pCAGGS-HA(control vector)and pCAGGS-HA-Pi-VP1 plasmids respectively.After 36 hours of transfection,Western blot was used to detect the expression of VP1 protein,and,the total RNA of two groups of cells was extracted for transcriptome sequencing(3 replicates in each group,6 samples in 2 groups).The obtained sequences were filtered and compared to obtain differentially-expressed genes,then the differentially-expressed genes were analyzed by qRT-PCR to verify the expression levels of key differentially expressed genes.[Results]RNA-Seq identified a total of 3376 differentially expressed genes(DEGs)|log2(Fold Change)|>0 and padj<0.05,including 1744 up-regulated genes and 1632 down-regulated genes.A large quantity of effector genes involved in immune response and inflammation are differentially expressed.The enrichment analysis of GO and KEGG showed that most of the differential gene GO entries were related to ribosomes,while the KEGG enrichment involved signal pathways related to natural immunity,such as TNF signal pathway and Toll receptor signal pathway.[Conclusion]After pCAGGS-HA-Pi-VP1 was transfected into PK-15 cells,the expression profile of the cells was changed.The analysis of DEGs and their function annotation information was helpful to understand the impact of Pi-VP1 protein on host cells,which provides basic data and theoretical support for the further studies of FMDV persistent infection.

关 键 词：转录组 基因表达谱 FMDV 持续感染 VP1 差异表达基因 

分 类 号：S852.65[农业科学—基础兽医学]