【作者】 王斌；

【导师】 陈薇； 王恒樑；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2012， 博士

【摘要】 甲型副伤寒沙门菌（SalmonellaParatyphiA，SPA）是甲型副伤寒（paratyphoidfeverA）的病原菌，通过粪－口途径传播，感染剂量为103～109个菌，能够引起伤寒样临床症状。人是唯一宿主。人群对甲型副伤寒沙门菌普遍易感，但儿童和青壮年发病最高。近些年来，在世界范围内，伤寒发病率有所降低，而甲型副伤寒发病率有明显升高。在我国，甲型副伤寒的发病率也相对较高，并在局部地区有爆发或流行。目前，旧的甲型副伤寒灭活疫苗由于副反应太大，已停止使用，尚无新的有效疫苗预防甲型副伤寒。因此对甲型副伤寒沙门菌的研究也越来越引起肠道致病菌领域学者的重视。当前分子生物学、基因组学、蛋白组学、生物信息学等快速发展的学科为疫苗的研发提供了革命性手段。通过对基因组信息的分析，筛选候选蛋白质抗原的方法被称为反向疫苗学（reversevaccinology），包括以下主要步骤：在获取病原体的基因组序列后，芯片分析预测编码分泌蛋白、表面相关抗原和毒力因子；蛋白质组学分析膜相关的蛋白；DNA微阵列鉴定高度表达的基因和体内上调的基因；从中综合分析可能的蛋白质抗原，将它们高通量克隆并表达；进行体内或体外免疫学实验分析，筛选有效的候选疫苗。可见，基因组序列和生物信息学分析的快速发展使基因注释不再是限定了总蛋白数和根据预测的功能分成几个组，而是预测细菌的可能全部蛋白在细胞中定位和功能等的信息。此外，蛋白质组学的发展也对疫苗研究起着积极的促进作用。蛋白质组学技术是对基因组学技术的补充，也是疫苗发展的有力技术支撑。综合基因组学、生物信息学、DNA微阵列和蛋白质组学技术，可以对存在于病原体的不同成分给予充分详细的定性、定量测定，对研发亚单位疫苗有很大的帮助。本研究所使用的甲型副伤寒沙门菌CMCC50973，是一株经筛选的毒力较强的地方流行株，计划用于甲型副伤寒亚单位疫苗的研究。为了全面了解其遗传信息，我们提取了细菌的基因组DNA，委托深圳华大公司测定了CMCC50973的全基因组序列。最终得到1个Scaffold环，全长为4,608,196bp，GC%为52.24%。预测基因4618个，tRNA100种，rRNA7种,sRNA56种。利用KEGG、COG、SwissProt、TrEMBL和NR数据库为基因进行了注释。CMCC50973的基因组通过与已完成测序的甲型副伤寒沙门菌ATCC9150的参考序列NC₀06511进行比较，一共找到了143个SNP位点，其中：在基因区的有112个；在基因间区的有31个，共找到12个InDel位点，其中插入位点6个，删除位点6个。该菌全基因组序列的获得，为进一步探寻基因间相互作用、新的调控因子等微生物更详尽的遗传学和生物学信息、预测和筛选新的更特异的保护性抗原基因，并在此基础上发展高效疫苗或经过遗传学操作改造疫苗菌株、构建活疫苗以及发展基因工程菌载体等打下了基础。表达蛋白质组学的研究是蛋白质组学研究的基础内容。建立起细胞在正常生理条件下的蛋白质参考图谱和数据库，首先能对细菌的蛋白质表达规律有整体的认识，为细菌的研究提供确切、宝贵的数据资料；其次能为后续的功能比较研究，如比较分析在变化了的条件下，蛋白质表达量的变化、翻译后的加工修饰、蛋白质在亚细胞水平上的改变等，提供很好的基础，从而发现和鉴定出特定功能的蛋白及其基因；第三是可以发现和解决一些生物学问题，如找到新的疫苗靶位，也为将来进行病原性研究、疫苗研制（免疫反应性抗原筛选）等相关工作提供重要的参考。因此，我们由地方流行株中筛选出来的毒力较强的菌株CMCC50973，进行了全菌蛋白表达谱的研究。本试验利用pH4.0-5.0、pH4.5-5.5、pH5.0-6.0、pH5.5-6.7的窄梯度胶条和pH6-11的碱性胶条，对CMCC50973对数生长末期的全菌体蛋白进行了双向电泳分离，然后对考马斯亮兰胶上可见的蛋白点取点进行胶内酶切，用MALDI-TOF/TOF-MS鉴定。共取蛋白点848个，鉴定到705个蛋白质点，代表519个基因编码产物，有多个蛋白点鉴定为同一基因产物的现象。所鉴定蛋白通过试验计算得出的分子量和等电点，与基因组注释中的预测值比较一致，但也有一些蛋白质理论值与计算值间存在一些差异。所鉴定蛋白质中多数功能是和能量代谢、营养素的运输和代谢相关；主要出现在糖酵解途径、三羧酸循环、磷酸戊糖途径、嘌呤和嘧啶的代谢等代谢通路中。在试验中鉴定到的67个蛋白被注释为“假想蛋白”（hypotheticalprotein），功能尚不明确。CMCC50973的全菌蛋白质组图谱的获得，帮助我们从整体层面了解了该菌基因组的蛋白表达信息，为研究细菌的致病机理和宿主特异性、基因组的表达情况、寻找新的疫苗靶位、以及免疫反应性抗原筛选等相关工作提供了基础。外膜蛋白（outermembraneprotein,OMP），是革兰氏阴性菌外膜的主要组成结构，在细菌的黏附、侵袭、耐药性产生等过程中都有着非常重要的作用。因此，在开发该类病原菌的疫苗过程中，对外膜蛋白的研究和筛选，已经成为各类疫苗研发过程中非常重要的内容。我们前期的试验证明甲型副伤寒沙门菌外膜蛋白BtuB与甲型副伤寒恢复期病人的血清有强烈的免疫反应，为了进一步研究该蛋白的免疫保护性，我们在大肠杆菌中克隆、表达了该蛋白，经纯化后获得了纯度约为90%的rBtuB蛋白，小鼠免疫显示重组蛋白具有良好的免疫原性。但对攻毒的保护率仅为60%，可考虑将其用于结合疫苗的载体，与其它抗原共同使用。更多还原

【Abstract】 Salmonella enterica serovar Paratyphi A （SPA）, a Gram-negative bacteria, is an emerging food and water-borne pathogen causing paratyphoid fever and is host-adapted to human only. The World Health Organization （WHO） has estimated that typhoid fever （including paratyphoid fever） causes approximately22million illnesses and220,000deaths per year. SPA is the most prevalent cause of paratyphoid fever. SPA infection rates have been rising, particularly in South East Asia including south of China, where this serovar is now responsible for30-50%of enteric fever cases. This increase has been associated with rises in antibiotic resistance among paratyphoid infections. It may also be associated with vaccination against Typhi, which unfortunately provides little cross-protection against Paratyphi A. Meanwhile, the present use of inactivated whole-cell paratyphoid A vaccines is restricted by their high rates of side effect. It is necessary to research and develop new protective vaccine of paratyphoid A fever. Therefore, the scholars of enteric pathogens field pay more attention to the study of SPA.At present, the rapid development of molecular biology, genomics, proteomics, bioinformatics and other subjects can provide the revolutionary methods for vaccine research and development. Reverse Vaccinology, an analysis based on the genomic information to screen candidate protein antigens, includes the following main steps: first, after obtaining the pathogen genome sequence, microarray analysis were used to predict encoding secreted proteins, surface-associated antigen and virulence factor; secondly, utility of proteomics to analyze the target proteins, such as outer membrane proteins, or DNA microarrays to identify highly expressed genes and genes regulation in vivo; thirdly, summarize the above results, the candidate protein antigens were defined and were high-throughput cloned and expressed in certain expression system; finally, candidate vaccine were selected by immunological experiments in vivo or in vitro. So, following the rapid development of genomics and bioinformatics analysis, gene annotation is no longer limited to the number of total proteins and function divided into several groups according to the forecast, but can forecast all proteins of cell including more information, such as localization and function. In addition, the development of proteomics plays an active role on vaccine research. Proteomics technology is a complement to genomics technology, also provides effective technical support on vaccine development. Comprehensive application of genomics, bioinformatics, DNA microarray and proteomics technology, can determine different components of the pathogens qualitatively and quantitatively. It is helpful for developing subunit vaccine.In this study, a reference strain of Salmonella enterica serovar Paratyphi A, CMCC50973, obtained from National Center for Medical Culture Collection （CMCC, Beijing, China）, was used. This strain is a stronger virulence isolates from a SPA-infected patient and was planned for the study of paratyphoid fever vaccine. The genomic DNA of CMCC50973strain was extracted and entrusted to BGI-Shenzhen to determine the complete genome sequence. The genome of CMCC50973consists of a single, circular chromosome of4,608,961bp with an average G+C content of52.24%, and including a total of4,618complete open reading frames （ORFs）,7rRNA clusters,100tRNAs,56sRNAs. All4,618genes were annotated according to different database of KEGG, COG, SwissProt, TrEMBL, or NR. Comparing with reference NC₀06511sequence of SPA strain ATCC9150, there are143single nucleotide polymorphisms （SNPs） and12small insertion or deletion events （Indels）. Of the143SNPs,112were located in open reading frames and31were in the intergenic regions. Out of12Indels, insertion events and deletion events have six respectively. In summary, the complete genome sequence of CMCC50973will lay the foundation to explore more detailed microbial genetics and biological information, such as the interaction between genes, the new regulatory factors, prediction and screening of new, more specific protective antigen gene, facilitate additional bioinformation and enable in-depth studies of SPA sequence variations.Expression proteomics research is the basis of proteomics research. To establish the protein reference map and database of whole-cell proteins under normal physiological conditions, first of all, can help us to have an overall understanding of the expression of protein; second, can provide a good foundation for subsequent comparative proteomics studies, such as comparative analysis the protein expression changes of different conditions of cell survival, post-translational processing and modification, protein changes at the subcellular level, also help for discovery and identification of the proteins with specific function and their genes; third, can help us to find some new vaccine targets, screen immune response antigens, and proceed future pathogenic studies. Therefore, we used the selected virulent SPA strain CMCC50973, performed the whole-cell protein expression profiling studies. In this study, the proteome reference maps of the end of cells growth log phase were thoroughly analyzed by the use of multiple overlapping narrow pH range （pH4.0-5.0, pH4.5-5.5, pH5.0-6.0, pH5.5-6.7, and pH6-11） two-dimensional gel electrophoresis. Altogether,705spots representing519different protein entries were identified by MALDI-TOF/TOF MS. Some gene products were found in multiple spots indicating isoforms. Calculated values of molecular weight and isoelectric point of identified proteins were similar to the predicted values of genome annotation, but there are also some differences. The most identified proteins contain which related to energy production and conversion, transport and metabolism of the three major nutrients, amino acid transport and translation, and so on. We also analyzed the abundance of identified proteins at different stages and confirmed the expression of67hypothetical proteins. CMCC50973whole-cell proteome map can help us understand the genomic expression information from the overall level, and provide a basis for looking for new vaccine targets or screening of the immune response antigen.Outer membrane proteins （OMPs） of Gram-negative bacteria play an essential role in bacterial pathogenesis and are directly involved in the interaction with host environments. Thus they represent important virulence factors and can induce a good immune response in host, which are usually facilitated the targets of antimicrobial drugs and vaccines. Salmonella OMPs also have been investigated as potential vaccine candidates and diagnostic antigens. Our former test showed that the outer membrane protein BtuB of SPA had a strong immune response with convalescent patient serum of paratyphoid fever. In order to further study the immunogenicity and immune protection of BtuB, the protein was cloned and expressed in Escherichia coli. About90%purity rBtuB protein was obtained after purification. Animal trials have shown that the recombinant protein had good immunogenicity. The lethal challenged experiments showed60%immunized mice with rBtuB were protected. The rBtuB protein showed good immunogenicity but unsatisfactory immune protection, it might be considered for conjugate vaccine carrier and used with other antigens.更多还原