基于表达谱芯片挖掘抗猪瘟病毒相关基因及其在不同猪瘟抗体水平下的表达差异
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摘要
本研究旨在筛选抗猪瘟病毒相关基因,并分析不同猪瘟抗体水平下这些相关基因在大白和长白猪中的表达差异。首先,基于表达谱芯片技术,采用Agilent猪4×44K全基因组表达谱芯片,分析4组试验材料(病毒模拟物PolyI:C转染的猪PK15细胞、甲基化酶抑制剂Aza-CdR转染的PK15细胞、PolyI:C及Aza-CdR共同转染的PK15细胞、无处理的mock细胞),获得试验组间显著性差异表达基因。进而对差异表达基因进行聚类分析和Gene Ontology(GO)分析。结果表明,存在显著差异表达的抗粘液病基因(MX1)和双链RNA依赖的蛋白激酶R基因(PKR)主要参与抗病毒相关的生物学过程。分析2个基因在不同猪瘟抗体水平下大白猪与长白猪外周血中的表达情况发现,2个基因在猪瘟高抗组中的表达量均显著高于未免疫组(P<0.05)。此外,MX1在猪瘟高抗组中的表达存在品种间差异,大白猪中的表达量极显著高于长白猪(P<0.01)。鉴于MX1和PKR基因都是干扰素通路中的重要基因,本研究结果提示,MX1和PKR基因可作为针对抗猪瘟病毒感染的重要候选基因。
The present study was conducted to screen the anti-CSF virus-related genes and to analyze the genes expression under the different CSF-antibody levels in Landrace and Yorkshire.Agilent porcine 4 X 44K microarray was used to analyze and obtain the differentially expressed genes in four research groups:virus mimic PolyI:C transfected cells,DNA methytransferase inhibitor Aza-CdR transfected PK15 cells,PolyI:C and Aza-CdR transfected cells,mock cells.The differentially expressed genes were analyzed by cluster and gene ontology(GO).The results showed that significantly differentially expressed genes of myxovirus resistant(MX1) and the double-stranded-RNA-dependent protein kinase(PKR) mainly enriched in the biological process of defense response to virus.The expression levels of both genes in peripheral blood of Landrace and Yorkshire were detected under different CSF-antibody levels.The expression levels of two genes were significantly higher in both breeds with high antibody levels than that with non-immune levels(P<0.05).In addition,MX1 was highly expressed in Yorkshire peripheral blood than that in Landrace(P<0.01).Considering MX1 and PKR are important genes in interferon pathway,the results indicate that both genes can be served as potential candidate genes for defending CSFV infection.
The present study was conducted to screen the anti-CSF virus-related genes and to analyze the genes expression under the different CSF-antibody levels in Landrace and Yorkshire.Agilent porcine 4 X 44K microarray was used to analyze and obtain the differentially expressed genes in four research groups:virus mimic PolyI:C transfected cells,DNA methytransferase inhibitor Aza-CdR transfected PK15 cells,PolyI:C and Aza-CdR transfected cells,mock cells.The differentially expressed genes were analyzed by cluster and gene ontology(GO).The results showed that significantly differentially expressed genes of myxovirus resistant(MX1) and the double-stranded-RNA-dependent protein kinase(PKR) mainly enriched in the biological process of defense response to virus.The expression levels of both genes in peripheral blood of Landrace and Yorkshire were detected under different CSF-antibody levels.The expression levels of two genes were significantly higher in both breeds with high antibody levels than that with non-immune levels(P<0.05).In addition,MX1 was highly expressed in Yorkshire peripheral blood than that in Landrace(P<0.01).Considering MX1 and PKR are important genes in interferon pathway,the results indicate that both genes can be served as potential candidate genes for defending CSFV infection.
引文
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[2]SUN J FJIANG Y,SHI Z X,et al.Proteomic alteration of PK-15cells after infection by classical swine fever virus[J].J Proteome Res,2008,7(12):5263-5269.
[3]LI Y,SHEN L,SUN Y,et al.A simplified serumneutralization test based on enhanced green fluorescent protein-tagged Classical swine fever virus[J].J Clin Microbiol,2013,51(8):2710-2712.
[4]周光炎.免疫学原理[M].上海:上海科学技术出版社,2008.
[5]ROTGER M,DANG K K,FELL AY J,et al.Genomewide mRNA expression correlates of viral control in CD4+T-cells from HIV-1-infected individuals[J].PLoS Pathogens,2010,6(2)sel000781.
[6]GLADUE D P,ZHU J,HOLINKA L G,et al.Patterns of gene expression in swine macrophages infected with classic swine fever virus detected by microarray[J].Virus Res,2010,151(1):10-18.
[7]LI L,XU Z,ZHOU Y,et al.Global effects of catecholamines on actinobacillus pleuropneumoniae gene expression[J].PLoS ONE,2012,7(2):e31121.
[8]TANG B.ZHAO R.SUN Y,et al.Interleukin-6 expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsRNA treatment[J].Mol Immunol,2011,48(8):1001-1008.
[9]OTT D,WUCHERT F,MURGOTT J,et al.The viral mimetic polyinosinic:polycytidylic acid(polyd:O)induces cellular responses in primary cultures from rat brain sites with an incomplete blood-brain barrier[J].Neurosci Lett,2012,530(1):64-68.
[10]CUI W Y.ZHAO S,POLANOWSKA-GRABOWSKA R,et al.Identification and characterization of Poly(I:C)-induced molecular responses attenuated by nicotine in mouse macrophages[J].Mol Pharmacol,2013,83(1):61-72.
[11]PATEL K,DICKSON J.DIN S,et al.Targeting of 5-aza-2-deoxycytidine residues by chromatin-associated DNMT1 induces proteasomal degradation of the free enzyme[J].Nucleic Acids Res,2010,38(13),4313-4324.
[12]FERNANDEZ G,ZEICHNER S.Cell line-dependent variability in HIV activation employing DNMT inhibitors[J].Virol 7,2010,7,266.
[13]KARPF A R,LASER A W,RIRIE T O,et al.Limited gene activation in tumor and normal epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine[J].Mol Pharmacol,2004,65:18-27.
[14]QIU X,HOTHER C,RALFKIAER U M,et al.Equitoxic doses of 5-azacytidine and 5-aza-2 deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome[J].PLoS ONE,2010,5(9):e12994.
[15]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25:402-408.
[16]ALLAN G.MEEHAN B.TODD D,et al.Novel porcine circovirus from pigs with wasting diseases syndrome[J].Vet Res,1998,142(17):467-468.
[17]温立斌,何孔旺,杨汉春,等.类圆环病毒因子P1感染对猪外周血单个核细胞抗病毒蛋白mRNA转录的影响[J].华北农学报,2010,25:7-11.
[18]赵协,张延虹,常维山.猪Mxl基因的克隆及在293T细胞中的表达[C].中国畜牧兽医学会畜牧兽医生物技术学分会暨中国免疫学会兽医免疫分会第八次学术研讨会论文集,2010:225-227.
[19]KOCHS G,HALLER O.Interfeton—induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids[J].Proc Natl Acad Sci USA,1999,96(5):2082-2086.
[20]吴圣龙,包文斌,鞠慧萍,等.猪Mx1基因第14外显子多态性分析及新突变位点的发现[J].遗传,2007,29(6):693-698.
[21]HORISBERGER M A.Virus-specific effects of recombinant porcine interferon-γand the induction of Mx proteins in pig cells[J].J Interferon Res,1992,12(6)-439-444.
[22]CHRISTOPHER L N,SIMPSON J,HALLER O,et al.Inhibition of a large double-stranded DNA virus by MxA protein[J].J Virol,2009,83(5):2310-2320.
[23]王波.仔猪猪瘟免疫程序的研究[D].泰安:山东农业大学,2009.
[24]吴家强,张学武,李俊,等.不同猪瘟免疫程序对仔猪母源抗体水平的影响[J].中国动物检疫,2010,27(11):46-48.
[25]阳凯.抗病毒信号通路调控新机制[D].上海:中国科学院上海生命科学研究院,2008.
[26]张正兴.猪瘟病毒慢性感染对细胞因子转录的影响[D].北京:中国兽医药品监察所,2012.
[27]FLORI L.GAILLARD C R.COCHET M,et al.Transcriptomic analysis of the dialogue between pseudorabies virus and porcine epithelial cells during infection[J].BMC Genomics,2008,9:123.
[28]LI Y T,ZHOU H B,WEN Z B,et al.Transcription analysis on response of swine lung to H1N1 swine influenza virus[J].BMC Genomics,2011,12:398.
[29]袁建琴,高斌战,梁新华.猪瘟病毒基因表达谱芯片的可靠性研究[J].激光生物学报,2008,17(5):613-619.
[30]王晓铄.dsRNA转染猪肾细胞后DNA甲基化抑制剂对全基因组DNA甲基化及表达的调控[D].北京:中国农业大学,2013.