【作者】 冯励；

【导师】 罗廷荣；

【作者基本信息】 广西大学 ， 微生物学， 2012， 博士

【摘要】 猪瘟病毒(Classical swine fever virus,CSFV)是一种带有囊膜的单股正链RNA病毒,与牛病毒性粘膜腹泻病毒和羊边界病毒同属于黄病毒科瘟病毒属。猪瘟病毒的基因组大小约12.3kb,只含有一个开放阅读框,编码一个由3898个氨基酸残基组成的多聚蛋白,该多聚蛋白在宿主细胞蛋白酶和病毒特异的蛋白酶共同作用下裂解为4个结构蛋白和8个非结构蛋白。家猪和野猪是该病毒的唯一宿主,猪瘟病毒引起的猪瘟是一种具有高发病率和高死亡率的烈性接触性传染病,给养猪业造成巨大的经济损失,世界卫生组织OIE将其列为A类必须申报的重要动物传染病之一,我国也将猪瘟定为一类动物传染病,并从2008年起列为动物疫病强制免疫项目。随着猪瘟疫苗的广泛使用,改变了猪瘟病毒的生态环境,猪瘟在流行特点和发病特点上发生了很大的变化,由大流行转变为地方性流行或散发,临床症状从典型转变为非典型,出现母猪繁殖障碍和新生仔猪先天感染。从精液、睾丸、扁桃体和淋巴结中检测到猪瘟病毒的存在,甚至在部分免疫了的猪体内检出猪瘟病毒,证实了猪瘟病毒存在持续感染现象。病毒要实现潜伏或持续性感染,必需逃脱宿主免疫系统的监视,突破宿主抗病毒防卫机制而获得免疫逃逸。研究猪瘟病毒与宿主之间的相互作用关系是理解病毒致病机制及其免疫应答的基础,猪瘟病毒在宿主体内或体外对靶细胞的影响正是它们相互作用的具体表现。PK-15细胞是猪瘟病毒体外感染的主要传代细胞,探索猪瘟病毒对PK-15细胞功能的影响,将有助于揭示猪瘟病毒感染的致病机制。因此,在本研究中我们应用real-time RT-PCR和Western blot方法对CSFV感染后宿主细胞的免疫应答基因表达的变化进行了研究,主要结果如下：1.建立了检测猪免疫应答基因mRNA表达水平的real-time RT-PCR方法。根据已知的猪源SLA-2、TAP1、SLA-DR、Ii、CD40、CD80、CD86、 IFN-α、IFN-β、 GAPDH基因和CSFV5’UTR基因片段的mRNA序列,采用生物软件Oligo6.0和Primer5.0辅助设计并合成了11对特异性引物；以Oligo(dT)18为引物,合成第一链cDNA;以cDNA为模板,分别进行11个目的基因片段的PCR扩增；胶回收PCR产物,把它们克隆到pMD18-T载体上并测序；对提取的重组质粒进行10倍梯度稀释作为标准模板进行PCR扩增,同时对荧光定量PCR反应的引物浓度和退火温度进行优化。应用11对引物分别扩增出目的基因目的条带,大小与预期片段相符合；real-time RT-PCR反应的最佳引物浓度是202.5nmol/μl,最佳退火温度是60℃；目的基因real-time RT-PCR融解曲线均显示单一溶解峰。结果表明,本试验成功地建立了检测PK-15细胞中SLA-2、TAP1、SLA-DR、Ii、CD40、CD80、 CD86、IFN-α、IFN-β、 GAPDH和CSFV5’UTR基因mRNA表达动态的实时荧光定量PCR方法。2.应用real-time RT-PCR方法检测猪瘟病毒感染后不同时间段的PK-15细胞中免疫应答基因的mRNA转录情况。在自然和实验环境下,猪瘟病毒能逃避宿主的免疫系统建立持续性感染。我们猜测,与MHC和细胞因子调控有关的一些基因卷入到免疫逃避事件中,但是具体的每一个基因在感染了CSFV后有什么变化尚不清楚。因此,我们用不同感染量猪瘟病毒Shimen毒株和GXXJ01毒株分别感染PK-15细胞,感染后1、3、12、24、36和48h应用real-time RT-PCR方法检测细胞中免疫应答基因SLA-2、TAP1、SLA-DR、Ii、CD40、CD80、CD86、IFN-α和IFN-β的mRNA表达动态。结果显示,SLA-2、TAP1、SLA-DR、Ii、CD40、 CD80和CD86的mRNA表达呈现明显的下调,其中SLA-DR和Ii的rnRNA下调特别显著,IFN-a和IFN-β的mRNA表达没有明显变化,表明CSFV可能参与了抑制免疫应答基因表达的机制。3.猪瘟病毒感染对宿主细胞中SLA-DR表达的影响。PK-15细胞感染猪瘟病毒Shimen毒株后,细胞内SLA-DR蛋白表达呈现明显的下调,在病毒感染后的24和36h检测不到SLA-DR蛋白,在感染后的48h检测到少量的SLA-DR蛋白。为了探讨猪瘟病毒诱导的SLA-DR的分子机制,从猪瘟病毒Shimen毒株中扩增出C、Npro和E2基因,将其与真核表达载体pcDNA3.0连接,成功构建了P-C、P-Npro和P-E2重组真核表达质粒。构建成功的3个真核表达质粒应用脂质体介导的转染方法进行体外细胞表达,转染后的1、3、12、24、36和48h检测细胞中SLA-DR的表达情况。结果显示,分别转染真核表达质粒P-E2和P-C后,PK-15细胞中SLA-DR的转录水平有所升高；转染真核表达质粒P-Npro后,PK-15细胞中SLA-DR的表达在3-24h显示升高,24h后开始下降,36h后恢复到与对照组一致的水平;在蛋白质水平对PK-15细胞中SLA-DR蛋白表达进行了检测,发现分别转染或共转染P-C、P-E2和P-C/P-E2后,细胞中SLA-DR蛋白的表达有比较明显的提高；转染P-C/P-Npro/P-E2后,细胞中SLA-DR蛋白的表达量稍有上升；分别转染或共转染P-Npro、P-C/P-Npro、P-Npro/P-E2后,PK-15细胞中SLA-DR蛋白的表达明显下降。表明猪瘟病毒E2和C两个基因单独或协同作用对宿主细胞SLA-DR的下调表达没有显著影响,Npro基因对PK-15细胞中SLA-DR的表达有一定的抑制作用。研究认为,病毒病的发生、发展是多基因、多步骤、多阶段参与的过程,猪瘟病毒的不同基因在抑制宿主细胞SLA-DR表达过程中的作用有待于进一步研究。更多还原

【Abstract】 Classical swine fever virus (CSFV), which is an enveloped, single-and positive-stranded RNA virus, with bovine viral diarrhea virus and border disease virus, belongs to the members of the Pestivirus genus within the family of Flaviviridae. The genome of CSFV is approximately12.3kb and contains a single large open reading frame encoding a polyprotein of3898amino acid, which undergoes co-and posttranslational processing and maturation into four structural and eight nonstructural proteins by viral proteases and host proteases. Domestic pigs and wild boars are the unique host of CSFV. CSFV causes a highly contagious infectious disease with high morbidity and mortality in pigs, resulting in substantial economic losses to the pig industry worldwide. Classical swine fever (CSF) is therefore classified as a List A disease by the Office International des Epizooties (OIE). In China, CSF is also listed as one of the severe animal epidemic disease and the State practices a system of immunization planning for execution of compulsory immunization against animal epidemics since2008.After implementation of strict immunization measures, ecological environment of CSFV were changed. CSF obviously presented changing in epidemiological forms and clinical signs. The large-scale CSF has rarely outbroken but sporadic epizootics still frequently occurred every year, and the clinical signs are dominated by typical to atypical forms of the disease, such as reproductive failures in pregnant sows and congenital infections in newborn piglets were observed. Virus is detected in semen, gonads, tonsil and lymph node even in certain vaccinated pigs, which confirmed CSFV have been established as persistent infection. Virus has to invade host defense mechanism and immune response and then establishes life-long, persistent infections. Therefore, the study on the interaction between host and CSFV is critical for understanding viral pathogenesis and immunity. For realizing the status of the cellular genes relative to immune response in vitro, PK-15cells were offered as a feasible model to investigate transcriptional changes during CSFV infection. The study may provide some useful data of insight into the interaction between CSFV and swine. In this study, real-time RT-PCR and Western blot approaches was applied to monitor the expression of immune response genes of host cells following CSFV infection. The results are mainly showed as follows. Experiment1. Development of the method for determination of several immune response genes mRNA levels in PK-15cells by real-time RT-PCR.According to the GenBank data, mRNA sequences of SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-a, IFN-p, GAPDH and5’-UTR of CSFV genes were downloaded, eleven pairs of specific primers were designed by biological Oligo6.0/Primer5.0software and synthesized. Oligo(dT)18were used as primer and first strand cDNA was synthesized from total RNA, which was isolated from swine lymphocytes. Using the synthesized cDNAs as template, eleven target fragments were amplified by polymerase chain reaction (PCR), respectively. The PCR products were electrophoresed on2%agarose gels and extracted. The extracted PCR products were cloned into pMD18-T Vector and sequenced. The extracted plasmids were diluted with series10-folds and were used as standard templates, and real-time PCRs were performed. In addition, the primer concentration and annealing temperature for each pair of specific primers was optimized, respectively. The results showed that each gene was amplified using specific primer pair. The optimal primer concentration was202.5nmol/μl and annealing temperatures for eleven primer pairs was60℃. The melting curve showed the unique peak for each PCR product, indicating that the method for determination SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-a, IFN-p and5’-UTR of CSFV mRNA levels in PK-15cells by real-time RT-PCR is successfully established.Experiment2. Transcriptional abundance of several immune response genes of PK-15cells following CSFV infection in vitro using real-time RT-PCR.CSFV evade the immune response and establish persistent infection under natural and experimental conditions. As we know, some genes relative to MHC and cytokine regulation are involved in persistent infection, but the extent of each gene responds to CSFV infection is unclear. In our study, nine immune response genes including SLA-2、TAP1、SLA-DR、Ii、CD40、CD80、CD86、 IFN-a and IFN-β were detected using real-time RT-PCR after the PK-15cells were infected with0.1Moi or1.0Moi Shimen or GXXJ01strain of CSFV, respectively. The results showed that these immune response genes basically presented down-regulation during CSFV infection, except IFN-a and IFN-β transcriptional levels of mRNA in CSFV-infected cells did not change significantly. The SLA-2, SLA-DR, and Ii genes were notably decreased. Our experiments concluded that CSFV maybe involved in the inhibition of immune response genes.Experiment3. Effects of classical swine fever virus infection on expression of SLA-DR gene in PK-15cells.Following Shimen strain of CSFV infection, the expression of SLA-DR presented down-regulation. SLA-DR is inhibited at24and36hpi and its expression restained in48hpi. In order to further exploration of the regulation between CSFV and SLA-DR, eukaryotic recombination expression plasmids P-C, P-Npro and P-E2were generated. The C, Npro and E2genes from Shimen strain of CSFV were cloned, then inserted into the expression vector pcDNA3.0. The eukaryotic recombination expression plasmids P-C, P-Npro and P-E2were constructed and transfected into PK-15cells with liposome infection protocol. The SLA-DR expression was detected by real-time RT-PCR and western blot. The results showed that SLA-DR mRNA expression showed a significantly up-regulation following P-C or P-E2transfection. SLA-DR presented up-regulation after transfection3to24hours, but in24hour SLA-DR become down-regulated, the SLA-DR mRNA expression was similar to controls in36and48hours. Western blot showed that SLA-DR expression presented an obviously up-regulation following P-C or P-E2transfection or P-C/P-E2co-transfection into PK-15cells. The co-transfection of three recombination vectors P-C/P-Npro/P-E2were carried out, SLA-DR expression presented slight up-regulated. However, SLA-DR expression showed an obviously down-regulation during P-Npro or P-C/P-Npro or P-Npro/P-E2co-transfection into PK-15cells. The results suggested that C or E2alone or synergistic did not showed significantly effect on the down-regulation of SLA-DR, Npro alone or synergistic showed slight effect on the down-regulation of SLA-DR. Current studies suggest that the incidence and development of virus disease is a multi-gene, multi-stage process participation, in which the relationship between CSFV and SLA-DR need to be further studied.更多还原