本文主要研究内容
作者张紫璇（2019）在《犬细小病毒YBYJ株全基因组克隆及其诱导MDCK细胞凋亡的初步研究》一文中研究指出：犬细小病毒(CPV)是能够引起犬出血性胃肠炎和心肌炎的高传染性和高致死性的疾病。通过粪-口传播,健康动物接触感染动物的粪便或者污染的表面获得病毒。犬作为伴侣动物携带CPV对其他动物造成潜在威胁。目前对于犬细小病毒病(CP)的预防主要依靠原始CPV-2毒株制备的疫苗,但由于病毒表面抗原易位容易导致免疫失败。CPV感染犬的分子机制尚不明确,有必要对CPV的致病机理进行深入的探讨。己知CPV可以引起宿主细胞产生病变,在体外可以诱导Hela,猫肾细胞F81和NLFK等细胞的凋亡,但这些细胞都不是伴侣动物犬的细胞,本文为探究CPV在体外是否可以诱导犬肾细胞系MDCK宿主细胞的凋亡展开试验。本试验首先根据GeneBank中已发表的CPV序列设计7对引物,对CPV YBYJ株全基因组分段扩增,克隆和测序,用DNAstar对测序结果拼接,其全基因组包含5 051个核苷酸。与GeneBank中发表的其它犬细小病毒毒株进行分子遗传进化学分析,结果表明,CPV YBYJ株与亚洲地区的毒株的亲缘关系最近,YBYJ 株与 CPV,SICHUAN(MH476590)的同源性最高,为 99%。与 GeneBank中发表的其它细小病毒毒株进行分子遗传进化学分析,结果表明,CPVYBYJ株与猫细小病毒(FPV)的亲缘关系最近,基因组同源性是98.6%,与马细小病毒(EqPV)的亲缘关系最远,基因组同源性仅为35.1%。本试验应用CPV YBYJ强毒株在体外感染MDCK宿主细胞,通过光学显微镜和荧光倒置显微镜观察到MDCK细胞被CPV感染后出现变圆、坏死和脱落等典型的细胞病变效应(CPE)。随着CPV感染时间延长,细胞活性逐渐降低,具有一定的时间依赖性。本试验成功建立CPV感染MDCK宿主细胞模型。通过细胞凋亡一DNA Ladder抽提试剂盒抽提CPV感染MDCK细胞的总DNA。琼脂糖凝胶电泳结果显示明显的DNA Ladder。荧光显微镜下用双色滤光片观察Annexin V-FITC/PI双染法染色的样品,CPV感染48 h时,检测孔内有较多细胞呈现明亮的苹果绿色,部分细胞呈现绿色胞膜包裹着红色的胞核。分别提取CPV诱导12 h,24 h,36h,48h和60h时MDCK细胞的蛋白,使用酶标仪确定Caspase-3相对活性,Caspase-3活性随着时间延长先升高后降低,并在48 h时达到峰值(p<0.01)。但随着时间继续延长,凋亡作用活性检测指标逐渐减弱。试验结果说明,CPV YBYJ强毒株在体外可以诱导MDCK宿主细胞发生凋亡,诱导作用在感染48 h时最明显,之后随着诱导时间延长,细胞趋于晚期凋亡并且坏死。本试验为后期研究CPV的分子生物学和致病机理奠定基础。
Abstract
quan xi xiao bing du (CPV)shi neng gou yin qi quan chu xie xing wei chang yan he xin ji yan de gao chuan ran xing he gao zhi si xing de ji bing 。tong guo fen -kou chuan bo ,jian kang dong wu jie chu gan ran dong wu de fen bian huo zhe wu ran de biao mian huo de bing du 。quan zuo wei ban lv dong wu xie dai CPVdui ji ta dong wu zao cheng qian zai wei xie 。mu qian dui yu quan xi xiao bing du bing (CP)de yu fang zhu yao yi kao yuan shi CPV-2du zhu zhi bei de yi miao ,dan you yu bing du biao mian kang yuan yi wei rong yi dao zhi mian yi shi bai 。CPVgan ran quan de fen zi ji zhi shang bu ming que ,you bi yao dui CPVde zhi bing ji li jin hang shen ru de tan tao 。ji zhi CPVke yi yin qi su zhu xi bao chan sheng bing bian ,zai ti wai ke yi you dao Hela,mao shen xi bao F81he NLFKdeng xi bao de diao wang ,dan zhe xie xi bao dou bu shi ban lv dong wu quan de xi bao ,ben wen wei tan jiu CPVzai ti wai shi fou ke yi you dao quan shen xi bao ji MDCKsu zhu xi bao de diao wang zhan kai shi yan 。ben shi yan shou xian gen ju GeneBankzhong yi fa biao de CPVxu lie she ji 7dui yin wu ,dui CPV YBYJzhu quan ji yin zu fen duan kuo zeng ,ke long he ce xu ,yong DNAstardui ce xu jie guo pin jie ,ji quan ji yin zu bao han 5 051ge he gan suan 。yu GeneBankzhong fa biao de ji ta quan xi xiao bing du du zhu jin hang fen zi wei chuan jin hua xue fen xi ,jie guo biao ming ,CPV YBYJzhu yu ya zhou de ou de du zhu de qin yuan guan ji zui jin ,YBYJ zhu yu CPV,SICHUAN(MH476590)de tong yuan xing zui gao ,wei 99%。yu GeneBankzhong fa biao de ji ta xi xiao bing du du zhu jin hang fen zi wei chuan jin hua xue fen xi ,jie guo biao ming ,CPVYBYJzhu yu mao xi xiao bing du (FPV)de qin yuan guan ji zui jin ,ji yin zu tong yuan xing shi 98.6%,yu ma xi xiao bing du (EqPV)de qin yuan guan ji zui yuan ,ji yin zu tong yuan xing jin wei 35.1%。ben shi yan ying yong CPV YBYJjiang du zhu zai ti wai gan ran MDCKsu zhu xi bao ,tong guo guang xue xian wei jing he ying guang dao zhi xian wei jing guan cha dao MDCKxi bao bei CPVgan ran hou chu xian bian yuan 、huai si he tuo la deng dian xing de xi bao bing bian xiao ying (CPE)。sui zhao CPVgan ran shi jian yan chang ,xi bao huo xing zhu jian jiang di ,ju you yi ding de shi jian yi lai xing 。ben shi yan cheng gong jian li CPVgan ran MDCKsu zhu xi bao mo xing 。tong guo xi bao diao wang yi DNA Ladderchou di shi ji he chou di CPVgan ran MDCKxi bao de zong DNA。qiong zhi tang ning jiao dian yong jie guo xian shi ming xian de DNA Ladder。ying guang xian wei jing xia yong shuang se lv guang pian guan cha Annexin V-FITC/PIshuang ran fa ran se de yang pin ,CPVgan ran 48 hshi ,jian ce kong nei you jiao duo xi bao cheng xian ming liang de ping guo lu se ,bu fen xi bao cheng xian lu se bao mo bao guo zhao gong se de bao he 。fen bie di qu CPVyou dao 12 h,24 h,36h,48hhe 60hshi MDCKxi bao de dan bai ,shi yong mei biao yi que ding Caspase-3xiang dui huo xing ,Caspase-3huo xing sui zhao shi jian yan chang xian sheng gao hou jiang di ,bing zai 48 hshi da dao feng zhi (p<0.01)。dan sui zhao shi jian ji xu yan chang ,diao wang zuo yong huo xing jian ce zhi biao zhu jian jian ruo 。shi yan jie guo shui ming ,CPV YBYJjiang du zhu zai ti wai ke yi you dao MDCKsu zhu xi bao fa sheng diao wang ,you dao zuo yong zai gan ran 48 hshi zui ming xian ,zhi hou sui zhao you dao shi jian yan chang ,xi bao qu yu wan ji diao wang bing ju huai si 。ben shi yan wei hou ji yan jiu CPVde fen zi sheng wu xue he zhi bing ji li dian ding ji chu 。
论文参考文献
论文详细介绍
论文作者分别是来自延边大学的张紫璇，发表于刊物延边大学2019-07-16论文，是一篇关于犬细小病毒株论文,全基因组论文,犬肾细胞论文,细胞凋亡论文,延边大学2019-07-16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，资料来自延边大学2019-07-16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：犬细小病毒株论文; 全基因组论文; 犬肾细胞论文; 细胞凋亡论文; 延边大学2019-07-16论文;