【作者】 史利军；

【导师】 章金刚； 陈焕春； 吴斌；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 西尼罗病毒(West Nile virus,WNV)是黄病毒科黄病毒属成员,最早于1937年于乌干达一位发热妇女的血液中分离到。西尼罗病毒引起的西尼罗脑炎和西尼罗热是严重影响人类及动物健康的传染病。WNV以往仅在非洲、西亚、中东地区流行,60年代初传入欧洲,自1999年起在西半球登陆,并相继在美国传播。美国CDC报道了美国2007年前半年WNV感染的情况,共有19个州的122人感染了WNV。WNV也可通过输血及器官移植传播。WNV的流行对人类及动物健康产生巨大的威胁。目前尚无针对WNV的特效治疗药物,亦无有效的疫苗预防WNV感染。我国目前还未见人感染WNV的病例和在动物体内发现WNV的报道。由于野生鸟类是其自然宿主,携带病毒的候鸟可能导致病毒在不同地区之间的传播,随着候鸟的迁徙及我国与世界其他国家之间的贸易、旅游、人员往来日益频繁,WNV通过各种途径传入我国的可能性很大。为有效应对我国可能发生的WNV的暴发流行,有必要开展对WNV检测方法的研究。本研究首先通过选取WNV较保守的衣壳蛋白基因区作为目的扩增片段,建立WNV荧光定量检测体系,同时对检测体系进行了方法学评价。对西尼罗病毒囊膜蛋白基因进行分析确定其抗原决定簇区域并克隆该囊膜蛋白结构域Ⅲ基因,进而通过基因重组技术获得病毒囊膜蛋白结构域Ⅲ抗原。基于表达的结构域Ⅲ抗原建立WNV抗体检测体系并对建立的检测体系进行了相应的方法学评价。WNV核酸及抗体检测体系的建立对于我国人与动物的WNV感染监控、人类输血安全以及技术储备等均具有重要的意义。主要分以下三个方面进行介绍。1.WNV核酸检测体系的建立及评价本研究建立了一种实时荧光定量PCR快速检测西尼罗病毒的方法。通过序列比对和blast分析,确定WNV衣壳蛋白保守区基因为检测的目的基因,引物采用Primer Premier5.0软件进行设计。本研究建立的检测方法利用SYBRGreenⅠ染料,相比探针法成本较低。通过熔解曲线分析表明,建立的检测方法在扩增过程中没有发现有二聚体的产生。本检测体系在用空白对照及类似的乙脑病毒作为扩增对照时,没有发现非特异性产物的生成,表明该体系对于WNV的检测是特异的。将阳性对照标准品进行10倍梯度稀释后,可检测到10~2copies/μL样品,表明该检测体系具有很高的检测灵敏度。通过6次批间重复检测,体系的变异系数小于3%,表明该检测体系具有良好的可重复性。同时也对检测体系进行了重复性、特异性及检测灵敏度的评价。总之建立的检测体系可以用于我国疑似WNV感染的人群、动物来源样品的检测,同时也可用于血液、血液制品等中可能带毒的样品的系统监测。2.西尼罗病毒E蛋白结构域Ⅲ在昆虫S2细胞中的表达与鉴定西尼罗病毒囊膜蛋白结构域Ⅲ(DomainⅢ)位于WNV病毒膜蛋白的最表面,是诱导机体产生抗体的主要保护性抗原,同时DomainⅢ也是WNV感染细胞时与细胞表面结合的主要位点。本研究根据GenBank中发表的WNV囊膜蛋白DomainⅢ基因序列设计了一对引物并分别引入NotⅠ和NcoⅠ酶切位点,用PCR方法扩增DomainⅢ基因片段。回收目的基因片段,并用NotⅠ和NcoⅠ限制性内切酶消化,经纯化后,将其定向克隆入果蝇表达载体pMT/B/V5中,构建重组表达载体pMT-DⅢ。测序验证正确后,用脂质体转染法与辅助质粒pCoBlast共转染果蝇S2细胞,通过Blasticidin(杀稻瘟素)进行加压筛选,获得了抗性细胞S2-DⅢ。提取S2-DⅢ细胞的基因组DNA,PCR法检测目的基因在果蝇S2细胞中的整合与表达。用终浓度500μmol/L的硫酸铜溶液诱导表达,收集无血清的细胞表达上清,样品浓缩后进行SDS-PAGE及Western Blotting检测。结果表明,本研究成功构建了重组表达载体pMT-DⅢ,转染细胞经12μg/mL的Blasticidin筛选及鉴定后获得了稳定表达WNV E DomainⅢ蛋白的果蝇S2细胞株。筛选的昆虫细胞重组表达DomainⅢ蛋白,经初步鉴定与预期相符,为建立WNV抗体ELISA检测体系提供了标准对照抗原。3.WNV抗体检测体系的建立与评价利用重组的西尼罗病毒囊膜蛋白结构域Ⅲ蛋白作为标准抗原建立其抗体检测体系。通过优化条件确立抗原的最佳包被浓度为2.5ug/mL,对照用抗体使用浓度为1600倍稀释。对建立的体系进行评价表明,该检测体系对WNV的检测特异性较高,且对其它类似病毒如乙脑病毒、牛病毒性腹泻病毒等的检测交叉反应性低。批内检测时平均值为1.002,标准差为0.0423,变异系数为0.0422。批间检测时平均值为1.162,标准差为0.151,变异系数为0.130。本研究基于表达的囊膜蛋白DⅢ蛋白建立了WNV抗体的检测技术,检测的各项指标均符合检测的要求。总之这一检测体系的建立将为我国研制WNV各种抗体检测试剂提供技术及方法上的参考。更多还原

【Abstract】 West Nile virus(WNV) is a member of the Japanese encephalitis (JE) virus group of the genus Flavivirus, family Flaviviridae. Infection of WNV is clearly an emerging and significant public health problem. It causes infectious disease in human and other animals featured by fever and encephalitis. The outbreak of WNV encephalitis in the United States in 1999 and subsequent epidemic in North America in the following years have caused great concerns. Moreover, with the development of the epidemic, there are more and more concerns on the transmitting routes in addition to mosquito bites, such as blood transfusion, organ transplantation and vertical transmission.Since there is no proven therapy for WN encephalitis in humans or animals, nor any human vaccine to prevent WNV infection, preventative public health measures are of primary importance in the control of transmission of WNV. Effective controlling transmission of WNV requires rapid and sensitive assaying systems.Real time quantitative PCR is used routinely for the high-throughput diagnosis of pathogens, such as WNV. In this study we developed and validated a less expensive assay with the intercalating dye SYBR green I . The SYBR green-based assay was as sensitive as the TaqMan assay for WNV.An indirect ELISA developed for assaying WNV antibody in infectious and suspicious samples from humans and animals. Detection of WNV antibody with ELISA has the advantage of a simple, rapid in vitro procedure, and the same time, the detecting method does not require the extensive support facilities needed for conventional methods of virus isolation. Another important aspect of this assay is the stability of the noninfectious viral protein, which is safety for assaying. Our research work includes the following:1. Establishment and Evaluation of Real-time PCR for Detection ofWNVA rapid real-time polymerase chain reaction (RT-PCR) for detection of WNV was established in this study. Primers were designed according to capsid protein gene with Primer Premier5.0. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green I . Amplifying curve showed that this method could successfully amplify WNV gene, nevertheless reference Japanese encephalitis and blank control were all negative. 10-fold dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assaying system could detect 10~2 Copies/μL WNV gene. The newly-built real-time PCR has high sensitivity, good specificity, reliable stability, so it has potential to apply in inspection and quarantine of WNV.2. Expression and Identification of WNV E DomainⅢin Drosophila S2 Cell LinesThe envelope protein is a major determinant of tropism and the primary target of virus-neutralizing antibody. In particular, domainIII of envelope is the major protective antigen of WNV, which can induce the production of neutralizing antibodies to neutralize the infectivity of the virus. This study constructed a expressed vector(pMT-DIII). pMT-DIII was transfected into S2 cells. S2- DIII cell which could permanently express recombinant DIII protein was established. The assessment of purity and speciality of WNV E DIII used SDS-PAGE and Western blotting.3. Establishment and Evaluation of Antibody ELISA Assaying System of WNVWe adapted an indirect Ab enzyme-linked assay to facilitate studies of WNV and evaluated its application. Serological diagnosis of WNV infection is complicated by extensive antigenic cross-reactivity with other closely related virus, such as JEV and BVDV. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WNV surveillance in our country. Here we describe a recombinant, S2 cell expressed antigen equivalent to structural domain III of the WNV envelope protein that has allowed clear discrimination of antibody responses to WNV from other related virus in indirect enzyme-linked assays using standardized control antiserum and some samples. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WNV surveillance in our country.更多还原