【作者】 王翔；

【导师】 杨泽晓；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2021， 硕士

【摘要】 兔瘟的学名为兔病毒性出血症(Rabbit viral hemorrhagic disease,RHD),是急性出血性疫病的一种,且对我国兔养殖业一直有着严重的威胁。该病侵害所有品种的穴兔(Oryctolagus cuniculus)且以全身脏器出血为特征,对家兔有着极高的感染率和致死率。目前对于RHDV致病机理的研究并不完善,为了进一步探究RHDV的致病机理,本文进行了如下研究:1.采用引物移步策略(primer-walking strategy)针对穴兔线粒体标准参考序列(NC＿001913.1)设计18对部分重叠的聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增引物,对线粒体基因组扩增,并且连接p CE2 TA/Blunt-Zero载体,转化入DH5α感受态细胞中,挑取阳性转化子进行测序鉴定并进行了RS17细胞系线粒体的全基因组拼接与分析,RS17线粒体基因组长度为17174 bp,注释到了38个特征。2.对RS 17细胞系线粒体基因组中的13个蛋白质编码基因(Protein-coding genes,PCGs)以及2个r RNA序列设计了q PCR引物并进行扩增,建立了一套RS17细胞系线粒体基因组的SYBR Green q PCR检测方法。每个靶基因针对各种模板不产生非特异扩增,15个基因的灵敏度结果:12S r RNA,16S r RNA的检出限为2.26和10.0 copies;COX1与COX2的检出限为13.40和10.90 copies;COX3与NADH3的检出限为16.50copies;NADH1、NADH2的检测限为16.26和17.28 copies;NADH4L、NADH4的检测限为2.24 copies;NADH5、NADH6和CYTB的检出限为2.20、2.02和12.70 copies;ATP8、ATP6的检出限为14.80 copies。3.将感染RHDV SCH18株死亡的实验兔肝脏用于纯化病毒的材料,使用蔗糖梯度密度离心纯化病毒后获得病毒颗粒,分别将病毒颗粒以及抽提的病毒颗粒RNA,用于RS17细胞系的感染,分不同时间点收获细胞,抽提总RNA并进行反转录后进行荧光定量检测。使用β-actin作为内参基因,用Pfaffl法分析线粒体基因组的转录水平。结果表明RHDV颗粒与细胞共培养使RS17细胞系线粒体基因组所有基因转录水平大幅度上调;RHDV RNA转染结果显示在转染初期所有的线粒体基因转录水平上调。为研究兔瘟致病机理奠定了基础。更多还原

【Abstract】 Rabbit viral hemorrhagic disease(RHD)is one of the acute contagious hemorrhagic diseases,and has been a great harm to the rabbit breeding industry in China.The disease infects all species of rabbits(Oryctolagus cuniculus)and is characterized by systemic viscera hemorrhage.It has a very high infection and fatality rate for rabbits.At present,the research on the pathogenic mechanism of RHDV is not perfect.In order to further explore the pathogenic mechanism of RHDV,the following studies were conducted in this research:1.A set of primers were designed by the primer-walking strategy based on the mitochondrial standard reference sequence(NC＿001913.1).The amplified product was conjugated with p CE2 TA/Blunt-Zero vector and transformed into DH5αcompetent cells.The positive transformants were selected and identified by sequencing,and the whole genome of RS17 cell line mitochondria was assembly and analyzed.The length of the mitochondrial genome of RS17 was 17174 bp,and 38features were annotated.2.A series of q PCR primers were designed and amplified for 13 protein-coding genes(PCGs)and 2 r RNA sequences in the mitochondrial genome of RS17.A set of SYBR Green q PCR detection method for RS17 mitochondrial genome was established.Each target gene did not produce nonspecific amplification against various templates.Sensitivity test of 15 genes:The detection limits of 12S r RNA and16S r RNA were 2.26 and 10.00 copies;COX1 and COX2 were 13.40 and 10.90copies;COX3 and NADH3 was 16.50 copies;NADH1 and NADH2 were 16.28 and17.28 copies;NADH4L and NADH4 was 2.24 copies;NADH5,NADH6 and CYTB were 2.20,2.02 and 12.70 ccopies;ATP8 and ATP6 was 14.80 copies.3.The dead rabbit liver infected with RHDV SCH18 strain was used as the raw material for virus purification.After the virus was purified by sucrose gradient density centrifugation,the virus particles were obtained.RS17 cell lines were infected with virus particles and transformed with RNA extracted from virus particles respectively,and the cells were harvested at different times.Then the cell total RNA was extracted and RT-q PCR carried out.Usingβ-actin as the reference gene,the transcription level of mitochondrial genome was analyzed by Pfaffl method.The results showed that the transcription level of all genes in the mitochondrial genome was significantly up-regulated in the co-culture of RHDV and cells.The results of RHDV RNA transfection showed that the transcription level of all mitochondrial genes was up-regulated in the early stage of transfection.It laid a foundation for studying the pathogenic mechanism of RHD.更多还原