影响猪流行性腹泻病毒复制的关键lncRNA筛选及其功能验证  

Screening and Functional Verification of Key lncRNA Affecting Porcine Epidemic Diarrhea Virus Replication

作　　者：杨荔 杜小妹 刘梦媛 吴圣龙[1] 包文斌[1] 吴正常[1] YANG Li;DU Xiaomei;LIU Mengyuan;WU Shenglong;BAO Wenbin;WU Zhengchang(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《中国畜牧兽医》2025年第2期792-800,共9页China Animal Husbandry & Veterinary Medicine

基　　金：江苏省研究生科研创新计划(KYCY23_3591);江苏省农业科技自主创新资金项目(CX(23)3084);校大学生科创基金(XCX20240786)。

摘　　要：[目的]探究长链非编码RNA(lncRNA)在猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)感染猪小肠上皮细胞(IPEC-J2)过程中的作用。[方法]利用感染复数(MOI)为1的PEDV感染IPEC-J2细胞构建细胞病变模型,通过RNA-Seq进行lncRNA测序,筛选出影响PEDV复制的关键lncRNA;利用实时荧光定量PCR和核质分离检测lncRNA表达和细胞定位;构建RNA干扰载体并转染IPEC-J2细胞,通过病毒拷贝数测定、Western blotting、半数组织培养感染剂量(TCID_(50))以及间接免疫荧光试验(IFA)检测lncRNA表达对PEDV复制水平的影响。[结果]PEDV感染IPEC-J2细胞24 h时,细胞病变最为明显,成功构建细胞病变模型。与对照组相比,PEDV感染组中存在61个差异表达lncRNA,其中19个上调,42个下调,筛选出1个显著上调且差异倍数较大的lncRNA——lncMSTRG.10733.2。实时荧光定量PCR结果显示,与对照组相比,PEDV感染IPEC-J2细胞后,lncMSTRG.10733.2表达水平显著上调(P<0.05)。核质分离测定结果显示,lncMSTRG.10733.2主要分布在IPEC-J2细胞质中。成功构建了lncMSTRG.10733.2瞬时干扰IPEC-J2细胞,干扰效率为45.9%。实时荧光定量PCR和Western blotting结果显示,lncRNA干扰后,PEDV-M基因mRNA相对表达量和PEDV N蛋白表达水平均显著升高(P<0.05)。TCID_(50)测定结果显示,lncRNA干扰后PEDV滴度极显著上升(P<0.01);IFA结果显示,lncRNA干扰后IPEC-J2细胞中的PEDV N蛋白荧光分布水平明显增加。[结论]本研究基于高通量测序从细胞水平上筛选鉴定出1个与PEDV感染密切相关的lncRNA,其表达水平上调有利于提高猪对PEDV感染的抵抗能力。研究结果揭示了lncRNA在PEDV感染宿主过程中的重要作用,为今后制定PEDV的抗病育种工作策略和筛选分子标记物奠定基础。[Objective]The experiment was to investigate the role of long non-coding RNA(lncRNA)in the infection of Porcine epidemic diarrhea virus(PEDV)in porcine small intestine epithelial cells(IPEC-J2).[Method]The cytopathic model was constructed using PEDV-infected IPEC-J2 cells with a multiplicity of infection(MOI)of 1.lncRNA sequencing was performed by RNA-Seq to screen out key lncRNA affecting PEDV replication.lncRNA expression and cell localization were detected by Real-time quantitative PCR and karyoplasmic separation.RNA interference vector was constructed and transfected with IPEC-J2 cells,then the effect of lncRNA expression on PEDV replication level was detected by virus copy number assay,Western blotting,50%tissue culture infectious dose(TCID 50)and indirect immunofluorescence assay(IFA).[Result]When PEDV infected IPEC-J2 cells for 24 h,the cytopathosis was most obvious,and the cytopathosis model was successfully constructed.Compared with control group,61 lncRNAs were differentially expressed in the PEDV infected group,among which 19 were up-regulated and 42 were down-regulated.lncRNA with significantly up-regulated and difference multiple was screened out—lncMSTRG.10733.2.Real-time quantitative PCR results showed that the expression of lncMSTRG.10733.2 was significantly up-regulated after PEDV infection with IPEC-J2 cells compared with control group(P<0.05).lncMSTRG.10733.2 was mainly distributed in the cytoplasm of IPEC-J2 cells.lncMSTRG.10733.2 transient interference IPEC-J2 cells were successfully constructed,and the interference efficiency was 45.9%.Real-time quantitative PCR and Western blotting results showed that the relative mRNA expression level of PEDV-M gene and PEDV N protein expression level were significantly increased after lncRNA interference(P<0.05).The results of TCID_(50) showed that the virus titer of PEDV increased extremely significantly after lncRNA interference(P<0.01).IFA results showed that the fluorescence distribution of PEDV N protein in IPEC-J2 cells was significantly increased

关 键 词：猪流行性腹泻病毒(PEDV) lncRNA IPEC-J2 病毒复制 

分 类 号：S852.651[农业科学—基础兽医学]