【作者】 王鹏；

【导师】 张建中；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2010， 博士

【摘要】 鼠疫（Plague）是一种由鼠疫耶尔森菌（Yersinia pestis）引起的自然疫源性疾病,是一个古老但迄今仍严重威胁人类的传染病。历史上,席卷全球的三次鼠疫大流行曾夺走了数亿人的生命；近年来,全球每年仍有千例左右的鼠疫病例报告,鼠疫仍是人类面临的一个重要公共卫生问题。鼠疫免疫学诊断是鼠疫监测及临床诊断的一种重要手段。鼠疫免疫学诊断主要是以鼠疫菌特异性抗原—F1荚膜抗原（F1）为基础。然而,由于自然界存在缺失F1的鼠疫菌株,这使单一针对F1的诊断方法存在漏诊的可能性；同时,F1又是一种温度调控表达的蛋白,在37℃大量表达,而低于28℃不表达或微量表达,这使现行检测方法应用于28℃以下生长鼠疫菌的免疫学检测受限。近年来,在浙江、广州等地血清学调查中,相继在一些正常人中检测到F1抗体,如何解释这一现象,对基于F1的检测方法提出质疑。本研究,一方面发展了以重组F1（rF1）为基础的免疫诊断方法；另一方面通过羟胺切割F1、克隆表达及化学合成F1片段等方法对F1的B细胞表位进行了分析,筛选出F1上的两个B细胞表位（"QFTTKVIGKDSRDFD"和TGSQDFFVRSIGSK"）,这两个B细胞表位可以作为判别交叉反应的诊断靶点。除F1外,本研究对21种鼠疫相关蛋白进行了克隆表达,通过对这些重组蛋白的免疫学评价,筛选出Caf1M和Pla两个潜在诊断靶点,并发展了以重组Caf1M（rCaf1M）和重组Pla （rPla）为基础的鼠疫诊断方法,为确诊是否与鼠疫感染相关提供了手段。利用上述鼠疫诊断方法,对浙江F1抗体阳性人血清进行了检测,结果显示,浙江F1抗体阳性人血清与F1上两个B细胞表位都发生反应,说明浙江F1抗体阳性人血清不是F1表位交叉反应引起；另外此血清与Caf1M和Pla两种鼠疫菌蛋白都发生反应,说明F1抗体阳性人血清与接触鼠疫菌相关。疫苗接种和自然感染都可以引发人群F1抗体阳性,为明确是那一种感染形式,需找出区分鼠疫疫苗株与自然分离株的生物标记。本研究选用鼠疫疫苗株EV76与云南自然分离株进行了比较蛋白质组与比较基因组学的分析。比较蛋白质组结果表明,两者之间未发现明显区别的蛋白标记,但得到两个有价值的发现。一是云南德宏地区鼠疫分离株较鼠疫疫苗株EV76及其它云南分离株高表达甘油激酶和硫酸盐转运蛋白；二是携带6Kb质粒（pYC）的云南鼠疫菌较鼠疫疫苗株EV76及其它云南鼠疫菌高表达GroEL伴侣蛋白。比较基因组学结果表明,除已知的鼠疫疫苗株EV76缺失102Kb pgm毒力岛基因外,鼠疫疫苗株EV76还缺失涉及细菌内甜菜碱的合成的3个相关基因（GI：4121108-4121110<NC₀08150>）及其它3个基因（GI:4121112-4121114<NC₀08150>）这些缺失基因可以作为潜在区别疫苗株与鼠疫自然分离株的靶点。总之,本研究筛选出F1上的两个B细胞表位及Caf1M和Pla两个潜在诊断靶点,采用这些靶点检测浙江F1抗体阳性人血清,提示其与接触鼠疫菌相关；同时通过比较基因组学分析发现,基因GI:4121108-4121110<NC₀08150>及GI:4121112-4121114<NC₀08150>是潜在区别鼠疫疫苗株与鼠疫自然分离株的靶点。更多还原

【Abstract】 Plague, a kind of natural focal disease caused by Yersinia pestis, is ancient but remains a serious threat to human so far. Historically, three global pandemics of plague deprived millions of lives of human beings. In recent years, there are still thousands of plague cases around the world. Plague is still a major public health problem for human being.Immunological diagnosis is an important mean in plague surveillance and clinical diagnosis. Immunological diagnosis of plague was based on detection of Fl specific antigen of Yersinia pestis. Strains with F1 antigen deficient exists in nature, so the current diagnostic methods targeting F1 antigen have the possibility of missed detection. Meanwhile, F1 antigen expression is controlled by temperature, with high expression at 37℃while micro or no expression under 28℃. The temperature-controlled expression of F1 antigen makes the current immunological diagnosis can not be used to strains living blow 28℃.In recent years, positive antibodies to F1 antigen were detected in some normal human in Zhejiang and Guangzhou province, China. It is also a challenge to current immunological diagnosis with F1 antigen.In this study, a immunological diagnosis was developed basing on recombinant F1 antigen （rF1）, and 2 B cell epitopes of F1 antigen were detected out by cutting F1 with hydroxylamine, cloning, expression and chemical synthesis of F1 fragments. They are "QFTTKVIGKDSRDFD" and "TGSQDFFVRSIGSK", which can be used to distinguish cross-reactions of F1 antigen.In addition to F1 antigen, the 21 related proteins of Yersinia pestis were cloned, and purified recombinant proteins were evaluated by anti-Yersinia pestis EV76 rabbit sera. Two potential diagnostic targets, CaflM and Pla were determined. And new diagnostic methods were developed with rCaf1M and rPla, which provide assistant means of plague diagnosis.Positive sera to F1 of Zhejiang normal human can react with rCaf1M, rPla and two B cell epitopes of F1 antigen. It indicates that the F1 antibodies may be associated with exposure of Yersinia pestis, vaccination or infection of Yersinia pestis.In order to find out biomarkers between vaccine strains and natural isolates of Yersinia pestis, proteome and genome comparison were performed between vaccine strain EV76 and Yunnan isolates of Yersinia pestis.No significant protein marker was found by proteome comparison, while there are two other valuable findings. The first, glycerol kinase and sulfate transport protein were highly expressed in Dehong region isolates comparing with vaccine strain EV76 and other Yunnan isolates. Second, GroEL chaperone protein were highly expressed in Yunnan isolates carrying 6Kb plasmid （pYC） comparing with vaccine strain EV76 and other Yunnan isolates,In addition to the 102Kb pgm pathogenicity island, we found 3 genes of betaine synthesis （GI:4121108-4121110<NC₀08150>） and 3 other genes （GI:4121112-4121114<NC₀08150>） were deficient in Yersinia pestis EV76 by comparative genomics. These genes could serve as potential biomarkers between vaccine strain EV76 and natural isolates of Yersinia pestis.In conclusion, two B cell epitopes of Fl antigen were confirmed in this study, and Caf1M and Pla were selected out as new diagnostic targets of plague. Positive sera of Zhejiang normal human to Fl can react with the above diagnostic targets. Moreover, the genes of GI:4121108-4121110<NC₀08150> and GI:4121112-41211104<NC₀08150> may be the potential biomarkers between vaccine strain EV76 and natural isolates of Yersinia pestis.更多还原