蛋白激酶C抑制剂及激活剂对鸭肠炎病毒增殖的影响
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摘要
为进一步探究鸭肠炎病毒(duck enteritis virus,DEV)的致病机理,试验采用鸭蛋白激酶C(protein kinase C,PKC)的抑制剂星形孢菌素(staurosporine,SP)和激活剂佛波醇(phorbol-12-myristate-13-acetate,PMA)研究PKC/PKCI能否对DEV的增殖产生影响,为阐述DEV致病机理提供新的思路。用SP/PMA处理正常鸭胚成纤维细胞(duck embryo fibroblast,DEF)后,实时荧光定量PCR方法检测不同时间段PKC/PKCI基因表达;用DEV病毒液感染SP/PMA处理的DEF后,收集不同时间段培养物,以Reed-Muench法计算DEV半数组织培养感染量(TCID50)值,实时荧光定量PCR方法检测分析DEV NP基因转录水平。结果显示,SP处理对宿主细胞PKC/PKCI基因表达水平无显著影响(P>0.05);而PMA处理可极显著提高宿主细胞PKC基因表达(P<0.01),但对PKCI基因表达水平无显著影响(P>0.05);SP/PMA处理对DEV复制能力影响显著(P<0.05;P<0.01),且在感染前期可显著或极显著降低DEV NP基因的转录水平(P<0.05;P<0.01)。综上所述,SP和PMA对PKC、PKCI基因表达水平的影响效果不同,且都能有效抑制DEV增殖,本试验结果可为DEV的防控及其致病机理的研究提供基础资料。
To identify the pathogenesis of duck enteritis virus(DEV),inhibitor and activator of PKC(SP and PMA)were used to research that whether PKC/PKCI could affect the proliferation of DEV,trying to provide some new ideas for understanding the mechanism of DEV infection.Normal duck fibroblasts(DEF)were treated with SP or PMA,and Real-time quantitative PCR method was used to measure the expression levels of PKCand PKCI genes at different time.The DEV was used to infect DEF which treated with SP or PMA,then the cell cultures were collected at different time,and the TCID50 and expression levels of DEV NPgene were measured by ReedMuench and Real-time quantitative PCR,respectively.The results showed that there was no significant effect of SP treatment on PKC/PKCI genes expression levels in DEF(P>0.05),whilethe PMA treatment could extremely significantly increased the PKCgene expression(P<0.01),but had no significant impact on PKCI gene(P>0.05);SP/PMA processing could significant or extremely significant affect the ability of DEV replication(P<0.05;P<0.01),and the expression level of DEV NPgene was significantly or extremely significant decreased at the early stage of infection(P<0.05;P<0.01).The above results indicated that the effects of SP and PMA on PKC/PKCI were different,while both of them could effectively inhibit the proliferation of DEV.These results could provide the basis for DEV prevention and its pathogenesis studying.
To identify the pathogenesis of duck enteritis virus(DEV),inhibitor and activator of PKC(SP and PMA)were used to research that whether PKC/PKCI could affect the proliferation of DEV,trying to provide some new ideas for understanding the mechanism of DEV infection.Normal duck fibroblasts(DEF)were treated with SP or PMA,and Real-time quantitative PCR method was used to measure the expression levels of PKCand PKCI genes at different time.The DEV was used to infect DEF which treated with SP or PMA,then the cell cultures were collected at different time,and the TCID50 and expression levels of DEV NPgene were measured by ReedMuench and Real-time quantitative PCR,respectively.The results showed that there was no significant effect of SP treatment on PKC/PKCI genes expression levels in DEF(P>0.05),whilethe PMA treatment could extremely significantly increased the PKCgene expression(P<0.01),but had no significant impact on PKCI gene(P>0.05);SP/PMA processing could significant or extremely significant affect the ability of DEV replication(P<0.05;P<0.01),and the expression level of DEV NPgene was significantly or extremely significant decreased at the early stage of infection(P<0.05;P<0.01).The above results indicated that the effects of SP and PMA on PKC/PKCI were different,while both of them could effectively inhibit the proliferation of DEV.These results could provide the basis for DEV prevention and its pathogenesis studying.
引文
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[11]杨颖,夏梦,殷俊磊,等.应用SYBR GreenⅠ荧光定量PCR方法检测人工感染雏鸭体内DEV NP基因表达水平[J].浙江农业学报,2011,23(4):703-707.YANG Y,XIA M,YIN J L,et al.Detection of virulent DEV NP gene in artificial infected ducklings using SYBR GreenⅠReal-time PCR[J].Acta Agriculturae Zhejiangensis,2011,23(4):703-707.(in Chinese)
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[13]刘俊麟,徐翔,崔志峰.以蛋白激酶C-θ为靶点的免疫抑制剂研究进展[J].中国生化药物杂志,2016,36(3):184-188.LIU J L,XU X,CUI Z F.Progresses in the research of immunosuppressor which take protein kinase C-θas target[J].Chinese Journal of Biochemical and Pharmaceuticals,2016,36(3):184-188.(in Chinese)
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[15]付更艳,贾号,赵庆孟,等.蛋白激酶C与免疫系统的关系及其抑制剂的研究进展[J].沈阳药科大学学报,2016,33(12):1001-1010.FU G Y,JIA H,ZHAO Q M,et al.Progress in the relationship between protein kinase C and immune system and its inhibitors[J].Journal of Shenyang Pharmaceutical University,2016,33(12):1001-1010.(in Chinese)
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[17]汪世雄,张军杰,叶昕.蛋白激酶抑制剂Flavopiridol对流感病毒复制的体外抑制作用[J].微生物学报,2012,52(9):1137-1142.WANG S X,ZHANG J J,YE X.Protein kinase inhibitor flavopiridol inhibits the replicationof influenza virus in vitro[J].Acta Microbiologica Sinica,2012,52(9):1137-1142.(in Chinese)
[18]关泽红,旭日干.细胞周期蛋白依赖性蛋白激酶2在单纯疱疹病毒复制中的作用[J].病毒学报,2008,2:96-100.GUAN Z H,XU R G.The role of cyclin-dependent protein kinase 2in the replication of herpes simplex virus[J].Chinese Journal of Virology,2008,2:96-100.(in Chinese)
[19]FRASER J E,WANG C,CHAN K W K,et al.Novel dengue virus inhibitor 4-HPR activates AT-F4independent of protein kinase R-like endoplasmic reticulum kinase and elevates levels of eIF2αphosphorylation in virus infected cells[J].Antiviral Research,2016,130:1-6.
[20]AHLQUIST P,NOUEIRY A O,LEE W M,et al.Host factors in positive-strand RNA virus genome replication[J].Journal of Virology,2003,77(15):8181-8186.
[21]JAKUBIEC A,TOURNIER V,DRUGEON G,et al.Phosphorylation of viral RNA dependent RNA polymerase and its role in replication of aplus-strand RNA virus[J].Journal of Biological Chemistry,2006,281(30):21236-21249.
[22]STORK J,PANAVIENE Z,NAGY P D.Inhibition of in vitro RNA binding and replicase activity by phosphorylation of the p33replication protein of cucumber necrosis tombusvirus[J].Virology,2005,343(1):79-92.
[23]DE I,FATA HARTLEY C,SAWICKI S G,et al.Functional analysis of ns P3phosphoprotein mutants of Sindbis virus[J].Journal of Virology,2003,77(24):13106-13116.
[2]刘情,邓伯雄,刘亚刚.鸭瘟病毒胸苷激酶基因的原核表达及其蛋白间接ELISA检测方法的建立[J].中国畜牧兽医,2015,42(12):3160-3166.LIU Q,DENG B X,LIU Y G.Prokaryotic expression of thymidine kinase gene of duck plague virus and establishment of an indirect ELISA based on the protein[J].China Animal Husbandry&Veterinary Medicine,2015,42(12):3160-3166.(in Chinese)
[3]刘荣昌,黄瑜,卢荣辉,等.鸭瘟病毒的分离鉴定及其UL2、TK基因序列分析[J].福建农业学报,2016,31(12):1257-1261.LIU R C,HUANG Y,LU R H,et al.Isolation and identification of duck plague virus and sequence analysis on its UL2and TK genes[J].Fujian Journal of Agricultural Sciences,2016,31(12):1257-1261.(in Chinese)
[4]温婷.鸭瘟病毒UL17蛋白与UL25蛋白、UL26.5蛋白的细胞共定位研究[D].雅安:四川农业大学,2014.WEN T.Co-localization of UL17protein with UL25protein,UL26.5protein in duck plague virus in infect cells[D].Ya'an:Sichuan Agricultural University,2014.(in Chinese)
[5]杨丽莎.鸭瘟病毒UL22基因的多抗制备、细胞内定位及真核表达研究[D].雅安:四川农业大学,2014.YANG L S.The eukaryotic expression,subcellular localization,polyclonal antibody preparation and molecular characterization analysis of duck plague virus UL22gene[D].Ya'an:Sichuan Agricultural University,2014.(in Chinese)
[6]张荷,毛君婷,杨颖,等.鸭肠炎病毒核衣壳蛋白受体的筛选与鉴定[J].病毒学报,2012,28(1):63-66.ZHANG H,MAO J T,YANG Y,et al.Screening and identification of receptor reacting with nucleocapsid protein of duck enteritis virus[J].Chinese Journal of Virology,2012,28(1):63-66.(in Chinese)
[7]GOULD C M,ANTAL C E,REYES G,et al.Active site inhibitors protect protein kinase C from dephosph-orylation and stabilize its mature form[J].Journal of Biological Chemistry,2011,286:28922-28930.
[8]程进.蛋白激酶C在肿瘤再增殖中的作用及作用机制研究[D].上海:上海交通大学,2014.CHENG J.Study on the role and mechanism of protein kinase C in tumor proliferation[D].Shanghai:Shanghai Jiao Tong University,2014.(in Chinese)
[9]罗乐,赵碧,郑敏,等.鸭源PKC基因荧光定量PCR检测方法的建立[J].西北农业学报,2016,25(3):342-346.LUO L,ZHAO B,ZHENG M,et al.Establishment of Real-time fluorescence quantitative PCR method for detecting PKC gene in duck[J].Acta Agriculturae Boreali-occidentalis Sinica,2016,25(3):342-346.(in Chinese)
[10]赵碧,熊朝丽,徐茂文,等.鸭肠炎病毒核衣壳蛋白质互作蛋白质基因荧光定量PCR检测方法的建立[J].江苏农业学报,2015,31(2):389-393.ZHAO B,XIONG C L,XU M W,et al.Establishment of a Real-time fluorescent quantitative PCR method for detecting the interacting protein(PKCI)gene of nucleocapsid protein of duck enteritis virus[J].Jiangsu Journal of Agricultural Sciences,2015,31(2):389-393.(in Chinese)
[11]杨颖,夏梦,殷俊磊,等.应用SYBR GreenⅠ荧光定量PCR方法检测人工感染雏鸭体内DEV NP基因表达水平[J].浙江农业学报,2011,23(4):703-707.YANG Y,XIA M,YIN J L,et al.Detection of virulent DEV NP gene in artificial infected ducklings using SYBR GreenⅠReal-time PCR[J].Acta Agriculturae Zhejiangensis,2011,23(4):703-707.(in Chinese)
[12]朱思薇,徐扬,李庆伟.蛋白激酶C-δ在细胞凋亡中的作用及其分子机制[J].中国生物化学与分子生物学报,2016,32(7):755-762.ZHU S W,XU Y,LI Q W.The function and mechanism of protein kinase C-δin apoptosis[J].Chinese Journal of Biochemistry and Molecular Biology,2016,32(7):755-762.(in Chinese)
[13]刘俊麟,徐翔,崔志峰.以蛋白激酶C-θ为靶点的免疫抑制剂研究进展[J].中国生化药物杂志,2016,36(3):184-188.LIU J L,XU X,CUI Z F.Progresses in the research of immunosuppressor which take protein kinase C-θas target[J].Chinese Journal of Biochemical and Pharmaceuticals,2016,36(3):184-188.(in Chinese)
[14]LIM P S,SUTTON C R,RAO S.Protein kinase C in the immune system:From signalling to chromatin regulation[J].Immunology,2015,146(4):508-522.
[15]付更艳,贾号,赵庆孟,等.蛋白激酶C与免疫系统的关系及其抑制剂的研究进展[J].沈阳药科大学学报,2016,33(12):1001-1010.FU G Y,JIA H,ZHAO Q M,et al.Progress in the relationship between protein kinase C and immune system and its inhibitors[J].Journal of Shenyang Pharmaceutical University,2016,33(12):1001-1010.(in Chinese)
[16]程靖华,孙英杰,陈鸿军,等.新城疫病毒ClassⅠ强毒株磷蛋白在细胞中的表达与定位[J].中国动物传染病学报,2013,21(1):17-22.CHENG J H,SUN Y J,CHEN H J,et al.Expression and locolzation analysis of phosphoprotein of a virulent strain from classⅠNewcastle disease virus[J].Chinese Journal of Animal Infectious Diseases,2013,21(1):17-22.(in Chinese)
[17]汪世雄,张军杰,叶昕.蛋白激酶抑制剂Flavopiridol对流感病毒复制的体外抑制作用[J].微生物学报,2012,52(9):1137-1142.WANG S X,ZHANG J J,YE X.Protein kinase inhibitor flavopiridol inhibits the replicationof influenza virus in vitro[J].Acta Microbiologica Sinica,2012,52(9):1137-1142.(in Chinese)
[18]关泽红,旭日干.细胞周期蛋白依赖性蛋白激酶2在单纯疱疹病毒复制中的作用[J].病毒学报,2008,2:96-100.GUAN Z H,XU R G.The role of cyclin-dependent protein kinase 2in the replication of herpes simplex virus[J].Chinese Journal of Virology,2008,2:96-100.(in Chinese)
[19]FRASER J E,WANG C,CHAN K W K,et al.Novel dengue virus inhibitor 4-HPR activates AT-F4independent of protein kinase R-like endoplasmic reticulum kinase and elevates levels of eIF2αphosphorylation in virus infected cells[J].Antiviral Research,2016,130:1-6.
[20]AHLQUIST P,NOUEIRY A O,LEE W M,et al.Host factors in positive-strand RNA virus genome replication[J].Journal of Virology,2003,77(15):8181-8186.
[21]JAKUBIEC A,TOURNIER V,DRUGEON G,et al.Phosphorylation of viral RNA dependent RNA polymerase and its role in replication of aplus-strand RNA virus[J].Journal of Biological Chemistry,2006,281(30):21236-21249.
[22]STORK J,PANAVIENE Z,NAGY P D.Inhibition of in vitro RNA binding and replicase activity by phosphorylation of the p33replication protein of cucumber necrosis tombusvirus[J].Virology,2005,343(1):79-92.
[23]DE I,FATA HARTLEY C,SAWICKI S G,et al.Functional analysis of ns P3phosphoprotein mutants of Sindbis virus[J].Journal of Virology,2003,77(24):13106-13116.