【作者】 江凌晓；

【导师】 俞守义；

【作者基本信息】 第一军医大学 ， 流行病学， 2004， 博士

【摘要】 鼠疫是一种严重危害人类健康的烈性自然疫源性传染病。历史上曾发生过三次鼠疫大流行，导致至少1.6亿人死亡。鼠疫的致病病原体为鼠疫耶尔森菌(Y.pestis)，属于肠杆菌科(Enterobacteraceae)耶尔森菌属(Yersinia)，该属现包括11个菌种，除鼠疫耶尔森菌、假结核耶尔森菌(Y.pseudotuberculosis)、小肠结肠炎耶尔森菌(Y.enterocolitica)对人致病外，其余8个菌种只对动物致病。 鼠疫主要经鼠蚤叮咬而传播，传染性强，病死率高，呈世界性分布，危害极大。近年来，全世界每年都有几千例病例报告，因此，2000年世界卫生组织将鼠疫列为重新抬头的传染病。1994年印度古吉拉特邦苏拉特市爆发鼠疫流行表明，即使是在现代公共卫生条件下，鼠疫仍有可能侵入大城市，造成严重的爆发流行。此外，鼠疫耶尔森菌还是传统的重要生物战剂，是国际恐怖分子可能用于制造生物恐怖的首选微生物之一。长期以来人们一直致力于鼠疫疫苗的研发，以对付这种自古以来就令人生畏的疾病。 当今世界曾经应用于人体免疫的鼠疫疫苗大体上可分为死菌苗、活菌苗两大类。最初的鼠疫死菌苗出自鼠疫正在肆虐的印度(1897)，而在越南战争(1961-1971)中得到较为广泛应用的鼠疫死菌苗为美军的USP疫苗。但近期的动物学研究表明，死菌苗对肺鼠疫的保护效果较差。鼠疫减毒活菌苗出现于1908年，在20世纪下半叶减毒的EV76活菌苗在许多国家广为使用。但由于EV76不是无毒株，在使用安全性方面存在许多隐患，因此许多国家已不再使用。到目前为止，尚无足够研究数据可以评价任何一种鼠疫疫苗的有效性和安全性，而且上述疫苗还伴有严重的副作用。所以发展一种安全、有效的鼠疫疫苗是当务之急。近年来，鼠疫疫苗的研发工作主要集中在新型减毒活疫苗株的选育和重组基因工程亚单位疫苗的研制上。 理论上讲，任何暴露于免疫系统的细菌抗原都有可能成为候选疫苗组分，但如果应用传统的研究手段一次只能对有限的几种抗原进行分析，而且如果体内和体外实验证实这几种抗原中没有候选疫苗组分的话就需要重新开始新一轮筛选，通常这又需要几年的时间。 在基因组时代，候选疫苗组分的筛选和鉴定一般分为以下4个步骤：(1)从基因组中选定一系列基因;(2)对选定基因进行大规模的克隆和表达; (3)对获得的重组蛋白进行纯化;(4)体内和体外分析，评价其免疫效果。目的基因的筛选是疫苗研发过程中的关键步骤之一，通常可能编码毒力因子、分泌性抗原、膜相关性抗原的基因可通过以下策略选定:(l)用生物信息学软件对全基因组序列进行比对后选定;(2)通过蛋白质组技术(如二维电泳技术、质谱技术)选定;(3)通过体内表达技术(in vivo exPressionteehnolo罗，wET)，条码标示诱变技术(signattire一tagged mutagenesis，STM)，DNA芯片技术选定。 蛋白质芯片分析技术是一种大规模、平行、快速的蛋白质分析技术平台，它可以同时对几千种蛋白质进行平行的分析。抗原芯片的研制，使同时研究机体对细菌全部蛋白质的体液免疫反应成为可能。这为候选疫苗的鉴定以及疾病的早期血清学诊断奠定了基础。因此利用蛋白质芯片技术，通过间接FLISA法就可以对感染血清中是否存在能与纯化抗原发生反应的组分进行鉴定。 本研究首先运用生物信息学技术，并结合鼠疫耶尔森菌基因组学和比较基因组学的初步研究结果，选择了57个可能编码外膜蛋白、粘附蛋白、侵袭性蛋白和对宿主细胞毒性的基因作为拟克隆和表达的目的基因。通过PCR技术、基因克隆和表达技术将上述目的基因克隆入表达载体PET32此L21中，获得了47个可以在大肠杆菌中正确表达目的蛋白的重组克隆子，其表达的目的蛋白占菌体总蛋白的30%一50%。目的蛋白经Ni~NTA纯化试剂盒纯化后纯度均大于85%。这些纯化的蛋白质作为探针被固定在玻片上制成鼠疫耶尔森菌致病相关蛋白芯片。 此芯片再与定期收集的经鼠疫耶尔森菌免疫的动物血清(新西兰兔、羊)或自然感染鼠疫动物(狗)血清进行FLISA反应，反应结果经扫读后进行数据分析。由此可实现对待测血清样品中鼠疫耶尔森菌致病相关蛋白相应抗体的平行检测，用以比较上述动物体内各种鼠疫耶尔森菌致病相关蛋白相应抗体出现的速度、滴度以及持续时间的差异。同时还可以比较上述各种致病相关蛋白在不同种属的实验动物中免疫性的差异。 研究结果显示，动物体内针对FI，o哪A(YPo1435)，LPp(Ypo2394)抗原的抗体反应较强，没有检测到针对Pla，Y叩T(YPCDI.20)，TyeA(YPeD 1 .35)，vi‘(钾eol.48)，YPoZ19o，钾03319等抗原的抗体反应。 一5-除了FI，omp^(YP01435)，LPp(YPo2394)以外，YopE(YPCDI.o6)，YopK(YpCD 1 .19)，YopD(YPCDI.28)，YoPN(YPCDI.39)，YopR (YPCDI.57)，Ymt(YPMTI.74)，YP01303，YP01411，YP02945也能刺激机体产生一定水平的抗体。 研究结果还表明重组蛋白Fl，o娜A(YPo 1435)，L即(钟02394)有可能用于鼠疫感染的血清学诊断。Y叩E(YPCD 1 .06)，Y叩K(YPCD 1 .19)，YopD(YPCD 1 .28)，YoPN(YpCDI.39)，YopR (YPCDI.57)，Ymt(Y更多还原

【Abstract】 Plague is an ancient disease which has caused over 160 million human deaths in the past and has not been eradicated from the modern world. The etiological agent of plague is Yersinia pestis. Y. pestis is one of ll species of the genus Yersinia. Three members of the genus are pathogenic to humans: Y.pestis, Y. pseudotuberculosis and Y. enterocolitica.Plague is usually contracted by human as a result of the bite of an infected flea. Each year there are several thousand reported cases of the disease world-wide and plague has been classified as a re-emerging disease by the World Health Organization. Endemic areas for the disease exist in China, Africa, Asia and South America. The Surat outbreak of plague in India in 1994 reminded the world of the threat of this horrible disease. Plague is an international public health risk and a notifiable disease. More over, Y. pestis has been of concern as one of the microorganisms which might be used illegitimately as a bioterrorisim agent against civilian or military communities.Because of the increasing plague casers and the likelihood of illegitimate use of Y. pestis, there is a requirement for an effective vaccine which can protect human against both bubonic and pneumonic plague.There are two kinds of vaccines have been used in human, live attenuated vaccine and killed whole cells vaccine (KWC) .The earliest report of a killed whole cell vaccine against plague was in 1897, and USP (a KWC vaccine) had been used in Vietnam during the period 1961-1971. But recent studies in animals have shown that this vaccine offers poor protection against pneumonic disease. Another vaccine, live attenuated vaccine ( EV76 strain) , has been in use since 1908, and was used hi many countries in the late 20 century. However, as EV76 is not fully avirulent, the safety of this vaccine in human is questionable. Today EV76 is considered unsuitable for humans in most countries. The efficacy and the safety of existing vaccines are not proven, and the use of these vaccines is known to be associated with adverse side effects.-7-Therefore there is an urgent need for an more efficacious plague vaccine.In recent years, efforts have focused on the development of a fully recombinant subunit vaccine for plague and the identification of a rationally attenuated mutant strain of Y. pestis.Theoretically speaking, any bacterial antigen exposed to the immune system represents a potential vaccine candidate. Anyway, only a few antigens can be analyzed simultaneously using conventional approach, and if negative results were obtained from in vitro and in vivo models, the process must repeated from the beginning, which often costs several years to get an ideal result.in the post-genomics era, three new strategies have been used to identify vaccine candidates. The three strategies share the same four basic steps: (1) selection of a set of genes from the whole genome; (2) high throughput cloning and expression of the selected genes; (3 ) purification of the recombinant proteins; and (4) in vitro and in vivo assays for identification of candidates.The virulence-associated genes selection is considered to be an important step in vaccine discovery. The three strategies differ in the way to select virulence-associated genes. According to the first strategy, genes are selected in silico utilizing algorithms that can predict genes encoding secreted and surface-associated antigens and virulence factors. These proteins are considered the most relevant for the induction of a protective immune response. Using the second approach, surface-exposed antigens are selected experimentally through the characterization of protein fractions using proteomics techniques such as two-dimensional gel electrophoresis (2D-GE) and mass spectrometry. The third approach is aimed at the identification of invasion and virulence-associated antigens and makes use of several new technologies including in vivo expression technology (IVET), signature-tagged mutagenesis ( STM) and DNA microarray technology.Protein microarray analysis offers a se更多还原