IFN-β表达与牛细小病毒VP1蛋白感染宿主细胞的机制研究  

Researches on Mechanism of Interferon-beta Expression and Bovine Parvovirus VP1 Protein Infection in the Host Cells

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作  者:魏锁成[1] 高恩玉 许玲珑 郭桢雅 裴梦源 袁肇方 WEI Suocheng;GAO Enyu;XU Linglong;GUO Zhenya;PEI Mengyuan;YUAN Zhaofang(College of Life Sciences and Engineering,Northwest Minzu University,Lanzhou 730030,China)

机构地区:[1]西北民族大学生命科学与工程学院,甘肃兰州730030

出  处:《西北民族大学学报(自然科学版)》2024年第4期31-40,共10页Journal of Northwest Minzu University(Natural Science)

基  金:甘肃省科技支撑计划项目(1604NKCA044);西北民族大学高原动物疾病创新团队建设项目(2020-88-07-3)。

摘  要:目的:系统研究IFN-β表达水平对牛细小病毒(BPV)VP1蛋白感染宿主细胞的作用与基础机制.方法:设计BPV Haden株(GenBank No:DQ335247)蛋白序列,提取其DNA,PCR扩增,构建BPV VP1和VP2真核表达载体,qRT-PCR检测HEK-293T细胞中VP1和VP2基因表达及细胞活力,Western blotting验证蛋白表达,检测拷贝数.用BPV VP1重组质粒转染HEK-293T细胞,提取RNA,qRT-PCR检测IFN-β水平以及Mx1、OAS1、ISG15、ISG56的mRNA水平、TBK1和IRF3转录水平.应用pCMV-Myc-BPV-VP1蛋白、TBK1、IRF3(5D)、MDA5和MAVS分别转染HEK-293T细胞,进行Western blotting和qRT-PCR,检测其表达水平.结果:对重组质粒进行双酶切鉴定,得到2022 bp和1611 bp的VP1和VP2片段,大小与预期相符;VP1和VP2 mRNA表达量比对照组分别增加5.5×10^(4)和7.4×10^(5)倍(P<0.001),BPV VP1组的病毒拷贝数比对照组增加5.8×10^(4)拷贝(P<0.001);BPV VP1显著抑制HEK-293T细胞中VSV介导的IFN-β产生;BPV VP1显著下调Mx、OAS、ISG15、ISG56表达水平,分别达30%、90%、84%和20%(P<0.01);TBK1和IRF3(5D)激活HEK-293T细胞中IFN-β产生,而且BPV VP1可使TBK1和IRF3转录水平下降81%和89%,使IFN-β生成量减少97%和90%.TBK1、IRF3(5D)、MDA5和MAVS均在HEK-293T细胞分布和表达,大小为84 kDa、55 kDa、118 kDa和58 kDa.转染pCMV-Myc-BPV-VP1蛋白后,TBK1、IRF3(5D)、MDA5和MAVS的表达水平均显著降低(P<0.001).结论:BPV VP1和VP2基因在HEK-293T细胞中高表达,BPV VP1蛋白显著促进BPV的体外增殖,并通过抑制ISGs转录水平,减少VSV介导的Mx、OAS、ISG15、ISG56转录水平.此外,显著抑制BK1和IRF3(5D)诱导的IFN-β生成,即BPV VP1蛋白通过IRF3或其通路降低IFN-I的抗病毒作用.pCMV-Myc-BPV-VP1能降低RLRs通路中TBK1、IRF3(5D)、MDA5和MAVS的表达水平.Objectives To explore systematically the interferon-beta(IFN-β)expression level effects and underlying mechanism following bovine parvovirus(BPV)VP1 protein infecting the host cells.Methods The protein sequence of BPV Haden strain(GenBank No:DQ335247)was designed.DNAs were extracted from the viruses and amplified with PCR.The eukaryotic expression vectors of BPV VP1 and VP2 were constructed.Expressions of VP1 and VP2 and cell viability were detected using qRT-PCR in HEK-293T cells.Western blotting was used to verify the protein expression levels and determine copy numbers of viruses.HEK-293T cells were transfected with BPV VP1 recombinant plasmid.IFN-βtranscription level,expression levels of Mx1,OAS1,ISG15,ISG56,TBK1 and IRF3 mRNAs were tested,respectively by applying qRT PCR.After transfection of HEK-293T cells with pCMV Myc BPV-VP1 protein,expression levels of TBK1,IRF3(5D),MDA5,and MAVS were evaluated respectively with both Western blotting and qRT PCR.Results VP1 and VP2 fragments about 2022 bp and 1611 bp were obtained by performing double enzyme digestion on the recombinant plasmid,which were consistent in the expectated size.The mRNA expression levels of VP1 and VP2 were increased by 5.5×10^(4) and 7.4×10^(5) folds as compared to the control group(P<0.001).Copy numbers of viruses in BPV VP1 treatment group were increased by 5.8×10^(4) as compared to the control group(P<0.001).BPV VP1 significantly inhibited IFN-βproduction in HEK-293T cells induced by vesicular stomatitis virus(VSV).BPV VP1 protein significantly downregulated the transcription levels of Mx,OAS,ISG15,and ISG56 with the decrement of 30%,90%,84%,and 20%,respectively(P<0.01).TBK1 and IRF3(5D)activated IFN-βproduction in HEK-293T cells,while BPV VP1 reduced their transcription levels of TBK1 and IRF3 by 81%and 89%,respectively.These decreased IFN-βproduction by 97%and 90%.All of TBK1,IRF3(5D),MDA5 and MAVS were distributed and expressed in HEK-293T cells,with sizes of 84 kDa,55 kDa,118 kDa and 58 kDa,respectively.After transfection of p

关 键 词:牛细小病毒 VP1蛋白 Ⅰ型干扰素 宿主细胞 

分 类 号:S852.65[农业科学—基础兽医学]

 

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