本文主要研究内容
作者韩青松（2019）在《鸡MDA5调控新城疫病毒免疫反应机制研究》一文中研究指出：新城疫（Newcastle Disease,ND）是由新城疫病毒（Newcastle Disease virus,NDV）强毒株引起的家禽急性高度接触性传染病,临床症状主要以体温升高、下痢、神经损伤、呼吸障碍、消化道和呼吸道的出血为特征。NDV可以感染包括家禽在内的至少240种鸟类和一些哺乳动物。该病在世界范围内广泛流行,给养禽业带来了巨大的经济损失。我国采取了积极免疫的措施进行ND的防控,已经控制了ND在我国大范围的流行,但由于我国养殖场生物安全体系差、NDV免疫失败等原因,ND的局部暴发时有发生。因此迫切需要探索新的ND防控策略。鸡MDA5（Chicken Melanoma Differentiation Association antigen 5,chMDA5）是鸡体细胞内监测外源病毒感染的模式识别受体（Pattern Recognition Receptor,PRR）之一,能够识别NDV感染,并介导干扰素（Interferon,IFN）的表达,启动鸡体的抗病毒天然免疫反应。探究chMDA5对NDV感染和宿主免疫反应的调控,有助于从机体角度阐明抗病毒和增强疫苗免疫力的机制,为揭示病毒和机体之间的相互作用提供新的资料,为提高NDV疫苗免疫效果提供新的参考。本课题主要研究内容和结果如下:1.chMDA5在体外诱导干扰素表达的研究NDV强毒株F48E9和弱毒株La Sota以0.1 MOI感染DF-1细胞,在感染后8 h、12 h、24 h和48 h用RT-qPCR方法检测chMDA5和IFN-β表达发现,F48E9和La Sota感染DF-1细胞都能引起chMDA5表达的上调,F48E9感染DF-1细胞诱导IFN-β表达上调,而La Sota感染DF-1细胞只有在感染后24 h时诱导IFN-β表达上调。将F48E9和La Sota以105PFU/只感染SPF鸡,用RT-qPCR方法检测chMDA5和IFN-β表达发现,F48E9和La Sota感染鸡体后均能诱导鸡体肺脏chMDA5和FN-β表达上调;F48E9感染鸡体后诱导鸡体脾脏chMDA5表达上调,诱导IFN-β表达先下调后上调再达到正常水平;La Sota感染鸡体后,12 h鸡体脾脏chMDA5表达下调,24 h和48 h后chMDA5表达上调,抑制鸡体脾脏IFN-β表达。应用带有chMDA5的真核表达质粒在DF-1上过表达chMDA5发现,在体外过表达chDMA5能够显著诱导细胞IFN-β表达,并抑制NDV强毒株F48E9的增殖。应用RNA干扰技术抑制DF-1细胞chMDA5表达发现,抑制细胞中chMDA5表达能够显著抑制细胞IFN-β表达,但对NDVF48E9的增殖无显著影响。2.诱导干扰素表达的chMDA5功能域研究chMDA5由1001个氨基酸残基组成。为了探究其诱导干扰素表达的功能域,根据chMDA5结构将其分为1328 aa、1483 aa、1642 aa、3251001 aa和全长等5个不同长度的片段,并以pCMV-3HA为骨架构建了5个表达这些chMDA5不同片段的真核表达载体。转染DF-1细胞,Western blot检测发现5个片段均能在DF-1细胞中得到表达。用RT-qPCR方法检测IFN-β表达,发现能诱导鸡IFN-β表达的片段都含有半胱氨酸蛋白酶激活和募集结构域（Caspase activation and recruitment domain,CARD）。1483 aa和1642 aa诱导IFN-β表达水平差异不显著,且和全长chMDA5诱导IFN-β表达水平差异不显著。因此,chMDA5的1483 aa可以作为研究chMDA5诱导IFN-β表达的功能域。3.chMDA5影响NDV对鸡致病性的研究构建并获得了能够表达chMDA5前483 aa的重组腺病毒rAd-MDA5和干扰chMDA5表达的重组腺病毒rAd-shM5。rAd-MDA5感染DF-1细胞后能够介导chMDA5（483 aa）的表达并诱导鸡IFN-β的表达;rAd-shM5感染DF-1细胞后能够显著降低chMDA5和IFN-β的表达,且下调F48E9感染细胞诱导的chMDA5和IFN-β的表达。rAd-MDA5注射鸡体后能够上调宿主chMDA5表达。与单独F48E9感染相比,共感染rAd-MDA5和F48E9能够显著上调F48E9攻毒后鸡体肺脏IFN-β的表达水平,降低肺脏病毒载量,并延长攻毒后鸡群的存活时间。而rAd-shM5注射鸡体后能够在鸡体内抑制chMDA5和IFN-β表达,但对F48E9感染后鸡群的存活时间无显著影响。4.chMDA5辅助激活适应性免疫反应的研究将表达chMDA5（483 aa）的重组腺病毒rAd-MDA5和NDV灭活疫苗共同免疫SPF鸡。免疫后每7天采集血液,用血凝抑制（Hemagglutination Inhibition,HI）方法检测血清抗体水平发现,腺病毒rAd-MDA5表达的chMDA5（483 aa）增强了NDV灭活疫苗诱导的体液免疫。在二免24 h后,采集鸡体脾脏,用RT-qPCR检测IL-4、IFN-γ和MHC-Ⅱ的表达,发现chMDA5（483 aa）增强了鸡体这些细胞免疫相关因子的表达。在鸡体攻毒前采集鸡体外周血,淋巴细胞增殖实验结果表明,chMDA5（483 aa）提高了联合免疫rAd-MDA5和NDV灭活疫苗鸡体的淋巴细胞刺激指数。将鸡体以104TCID50/只的剂量感染NDV强毒株F48E9发现,联合免疫rAd-MDA5和NDV灭活疫苗,降低了感染引起的发病率、组织损伤、器官病毒载量和排毒。这些结果表明,chMDA（483 aa）增强了NDV灭活疫苗的免疫效果。综上所述,本研究详细阐述了NDV感染细胞和鸡体后chMDA5和IFN-β的表达规律,研究了chMDA5诱导IFN表达的功能域,阐明了chMDA5对NDV增殖及其对鸡致病性的影响,揭示了chMDA5（483 aa）增强NDV灭活疫苗免疫效果的机制。一方面为进一步研究chMDA5功能提供理论基础,为进一步研究chMDA5帮助宿主抵抗NDV的感染提供依据;另一方面为增强NDV疫苗免疫效果提供了新的方法,也为ND新型疫苗开发提供了候选佐剂。
Abstract
xin cheng yi （Newcastle Disease,ND）shi you xin cheng yi bing du （Newcastle Disease virus,NDV）jiang du zhu yin qi de jia qin ji xing gao du jie chu xing chuan ran bing ,lin chuang zheng zhuang zhu yao yi ti wen sheng gao 、xia li 、shen jing sun shang 、hu xi zhang ai 、xiao hua dao he hu xi dao de chu xie wei te zheng 。NDVke yi gan ran bao gua jia qin zai nei de zhi shao 240chong diao lei he yi xie bu ru dong wu 。gai bing zai shi jie fan wei nei an fan liu hang ,gei yang qin ye dai lai le ju da de jing ji sun shi 。wo guo cai qu le ji ji mian yi de cuo shi jin hang NDde fang kong ,yi jing kong zhi le NDzai wo guo da fan wei de liu hang ,dan you yu wo guo yang shi chang sheng wu an quan ti ji cha 、NDVmian yi shi bai deng yuan yin ,NDde ju bu bao fa shi you fa sheng 。yin ci pai qie xu yao tan suo xin de NDfang kong ce lve 。ji MDA5（Chicken Melanoma Differentiation Association antigen 5,chMDA5）shi ji ti xi bao nei jian ce wai yuan bing du gan ran de mo shi shi bie shou ti （Pattern Recognition Receptor,PRR）zhi yi ,neng gou shi bie NDVgan ran ,bing jie dao gan rao su （Interferon,IFN）de biao da ,qi dong ji ti de kang bing du tian ran mian yi fan ying 。tan jiu chMDA5dui NDVgan ran he su zhu mian yi fan ying de diao kong ,you zhu yu cong ji ti jiao du chan ming kang bing du he zeng jiang yi miao mian yi li de ji zhi ,wei jie shi bing du he ji ti zhi jian de xiang hu zuo yong di gong xin de zi liao ,wei di gao NDVyi miao mian yi xiao guo di gong xin de can kao 。ben ke ti zhu yao yan jiu nei rong he jie guo ru xia :1.chMDA5zai ti wai you dao gan rao su biao da de yan jiu NDVjiang du zhu F48E9he ruo du zhu La Sotayi 0.1 MOIgan ran DF-1xi bao ,zai gan ran hou 8 h、12 h、24 hhe 48 hyong RT-qPCRfang fa jian ce chMDA5he IFN-βbiao da fa xian ,F48E9he La Sotagan ran DF-1xi bao dou neng yin qi chMDA5biao da de shang diao ,F48E9gan ran DF-1xi bao you dao IFN-βbiao da shang diao ,er La Sotagan ran DF-1xi bao zhi you zai gan ran hou 24 hshi you dao IFN-βbiao da shang diao 。jiang F48E9he La Sotayi 105PFU/zhi gan ran SPFji ,yong RT-qPCRfang fa jian ce chMDA5he IFN-βbiao da fa xian ,F48E9he La Sotagan ran ji ti hou jun neng you dao ji ti fei zang chMDA5he FN-βbiao da shang diao ;F48E9gan ran ji ti hou you dao ji ti pi zang chMDA5biao da shang diao ,you dao IFN-βbiao da xian xia diao hou shang diao zai da dao zheng chang shui ping ;La Sotagan ran ji ti hou ,12 hji ti pi zang chMDA5biao da xia diao ,24 hhe 48 hhou chMDA5biao da shang diao ,yi zhi ji ti pi zang IFN-βbiao da 。ying yong dai you chMDA5de zhen he biao da zhi li zai DF-1shang guo biao da chMDA5fa xian ,zai ti wai guo biao da chDMA5neng gou xian zhe you dao xi bao IFN-βbiao da ,bing yi zhi NDVjiang du zhu F48E9de zeng shi 。ying yong RNAgan rao ji shu yi zhi DF-1xi bao chMDA5biao da fa xian ,yi zhi xi bao zhong chMDA5biao da neng gou xian zhe yi zhi xi bao IFN-βbiao da ,dan dui NDVF48E9de zeng shi mo xian zhe ying xiang 。2.you dao gan rao su biao da de chMDA5gong neng yu yan jiu chMDA5you 1001ge an ji suan can ji zu cheng 。wei le tan jiu ji you dao gan rao su biao da de gong neng yu ,gen ju chMDA5jie gou jiang ji fen wei 1328 aa、1483 aa、1642 aa、3251001 aahe quan chang deng 5ge bu tong chang du de pian duan ,bing yi pCMV-3HAwei gu jia gou jian le 5ge biao da zhe xie chMDA5bu tong pian duan de zhen he biao da zai ti 。zhuai ran DF-1xi bao ,Western blotjian ce fa xian 5ge pian duan jun neng zai DF-1xi bao zhong de dao biao da 。yong RT-qPCRfang fa jian ce IFN-βbiao da ,fa xian neng you dao ji IFN-βbiao da de pian duan dou han you ban guang an suan dan bai mei ji huo he mu ji jie gou yu （Caspase activation and recruitment domain,CARD）。1483 aahe 1642 aayou dao IFN-βbiao da shui ping cha yi bu xian zhe ,ju he quan chang chMDA5you dao IFN-βbiao da shui ping cha yi bu xian zhe 。yin ci ,chMDA5de 1483 aake yi zuo wei yan jiu chMDA5you dao IFN-βbiao da de gong neng yu 。3.chMDA5ying xiang NDVdui ji zhi bing xing de yan jiu gou jian bing huo de le neng gou biao da chMDA5qian 483 aade chong zu xian bing du rAd-MDA5he gan rao chMDA5biao da de chong zu xian bing du rAd-shM5。rAd-MDA5gan ran DF-1xi bao hou neng gou jie dao chMDA5（483 aa）de biao da bing you dao ji IFN-βde biao da ;rAd-shM5gan ran DF-1xi bao hou neng gou xian zhe jiang di chMDA5he IFN-βde biao da ,ju xia diao F48E9gan ran xi bao you dao de chMDA5he IFN-βde biao da 。rAd-MDA5zhu she ji ti hou neng gou shang diao su zhu chMDA5biao da 。yu chan du F48E9gan ran xiang bi ,gong gan ran rAd-MDA5he F48E9neng gou xian zhe shang diao F48E9gong du hou ji ti fei zang IFN-βde biao da shui ping ,jiang di fei zang bing du zai liang ,bing yan chang gong du hou ji qun de cun huo shi jian 。er rAd-shM5zhu she ji ti hou neng gou zai ji ti nei yi zhi chMDA5he IFN-βbiao da ,dan dui F48E9gan ran hou ji qun de cun huo shi jian mo xian zhe ying xiang 。4.chMDA5fu zhu ji huo kuo ying xing mian yi fan ying de yan jiu jiang biao da chMDA5（483 aa）de chong zu xian bing du rAd-MDA5he NDVmie huo yi miao gong tong mian yi SPFji 。mian yi hou mei 7tian cai ji xie ye ,yong xie ning yi zhi （Hemagglutination Inhibition,HI）fang fa jian ce xie qing kang ti shui ping fa xian ,xian bing du rAd-MDA5biao da de chMDA5（483 aa）zeng jiang le NDVmie huo yi miao you dao de ti ye mian yi 。zai er mian 24 hhou ,cai ji ji ti pi zang ,yong RT-qPCRjian ce IL-4、IFN-γhe MHC-Ⅱde biao da ,fa xian chMDA5（483 aa）zeng jiang le ji ti zhe xie xi bao mian yi xiang guan yin zi de biao da 。zai ji ti gong du qian cai ji ji ti wai zhou xie ,lin ba xi bao zeng shi shi yan jie guo biao ming ,chMDA5（483 aa）di gao le lian ge mian yi rAd-MDA5he NDVmie huo yi miao ji ti de lin ba xi bao ci ji zhi shu 。jiang ji ti yi 104TCID50/zhi de ji liang gan ran NDVjiang du zhu F48E9fa xian ,lian ge mian yi rAd-MDA5he NDVmie huo yi miao ,jiang di le gan ran yin qi de fa bing lv 、zu zhi sun shang 、qi guan bing du zai liang he pai du 。zhe xie jie guo biao ming ,chMDA（483 aa）zeng jiang le NDVmie huo yi miao de mian yi xiao guo 。zeng shang suo shu ,ben yan jiu xiang xi chan shu le NDVgan ran xi bao he ji ti hou chMDA5he IFN-βde biao da gui lv ,yan jiu le chMDA5you dao IFNbiao da de gong neng yu ,chan ming le chMDA5dui NDVzeng shi ji ji dui ji zhi bing xing de ying xiang ,jie shi le chMDA5（483 aa）zeng jiang NDVmie huo yi miao mian yi xiao guo de ji zhi 。yi fang mian wei jin yi bu yan jiu chMDA5gong neng di gong li lun ji chu ,wei jin yi bu yan jiu chMDA5bang zhu su zhu di kang NDVde gan ran di gong yi ju ;ling yi fang mian wei zeng jiang NDVyi miao mian yi xiao guo di gong le xin de fang fa ,ye wei NDxin xing yi miao kai fa di gong le hou shua zuo ji 。
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论文作者分别是来自西北农林科技大学的韩青松，发表于刊物西北农林科技大学2019-07-11论文，是一篇关于新城疫病毒论文,腺病毒载体论文,免疫反应论文,西北农林科技大学2019-07-11论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自西北农林科技大学2019-07-11论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：新城疫病毒论文; 腺病毒载体论文; 免疫反应论文; 西北农林科技大学2019-07-11论文;