甲型副伤寒沙门菌CMCC 50973的基因表达谱及外膜蛋白BtuB的保护性研究

发布时间：2018-01-24 14:53

本文关键词： 甲型副伤寒沙门菌基因组学蛋白质组学疫苗外膜蛋白 BtuB蛋白　出处：《中国人民解放军军事医学科学院》2012年博士论文　论文类型：学位论文

【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NC_006511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。
[Abstract]:Salmonella paratyphi A (SalmonellaParatyphiA, SPA) is paratyphi A (paratyphoidfeverA) pathogen spread through the fecal oral route, infection dose of 103 ~ 109 bacteria, can cause typhoid symptoms. The human is the only host. People of paratyphoid salmonella generally susceptible, but children and youth the highest incidence of adults. In recent years, in the world, the incidence of typhoid and paratyphoid fever has decreased, the incidence rate increased significantly. In our country, the incidence of paratyphoid fever is relatively high, and in the local area or flow. The outbreak of the old paratyphoid vaccine due to side the reaction is too large, has been discontinued, there is no effective vaccine to prevent new paratyphoid fever. So the study of Salmonella paratyphi has attracted more and more scholars pay more attention to the field of intestinal bacteria.
The current molecular biology, genomics, proteomics, bioinformatics provides a revolutionary and rapid developing discipline for vaccine development. Through the analysis of genomic information, method for screening candidate protein antigen is known as reverse vaccinology (reversevaccinology), includes the following steps: after obtaining the pathogen genome sequence. Chip analysis and prediction encoding secreted protein, surface antigen and virulence factor; proteomic analysis of membrane associated protein; gene and up regulate the DNA microarray to identify highly expressed genes; from the comprehensive analysis of possible protein antigens, their high-throughput cloning and expression; analysis of in vivo or in vitro immunological experiments, screening of candidate vaccine effective. Obviously, the rapid development of genome sequence analysis and bioinformatics that gene annotation is no longer limited the total number of protein And divided into several groups according to the prediction function, but all prediction protein located in the cell and function of bacterial information. In addition, the development of proteomics for vaccine research plays a positive role in promoting. Proteomics is complementary to genomics technology, but also a powerful technical support for vaccine development. Synthetic Genomics, bioinformatics, DNA microarray and proteomics technology can give qualitative, fully detailed on different components exist in the quantitative determination of pathogens, are of great help for the development of subunit vaccine.
This study uses Salmonella paratyphi CMCC50973, a strain by screening strong virulent isolates, plan for subunit vaccine research of paratyphoid fever. In order to fully understand their genetic information, we extracted genomic DNA of bacteria, the whole genome sequence of CMCC50973 was commissioned by Shenzhen Hua company. 1 Scaffold ring, 4608196bp in length, GC% 52.24%. predicts 4618 genes, tRNA100, rRNA7, sRNA56. Using KEGG, COG, SwissProt, TrEMBL and NR of.CMCC50973 genome annotation database through the comparison with the reference sequence of NC_006511 has completed the sequencing of Salmonella paratyphi A ATCC9150 gene. Found a total of 143 SNP loci, including: in the gene region of 112; in the intergenic region of the 31, found a total of 12 InDel loci, the insertion site 6, delete the site 6 A. Get the whole genome sequence of the bacteria, to further explore the interaction between genes, genetics and biological information regulator of microorganisms such as new and more detailed, predicting and screening of protective antigens of more specific gene, and on the basis of the development of efficient vaccines through genetic manipulation or transformation of vaccine strains, construction of live vaccine as well as the development of genetically engineered bacteria carrier to lay the foundation.
Study on the expression of proteomics is the proteomics research foundation. Establish cells in normal physiological conditions of the protein reference map and database, the first rule of bacterial protein expression can have an overall understanding, provide exact bacteria of valuable data; secondly to study for the following function. As a comparative analysis in the changed conditions, the changes of the expression of protein, posttranslational processing, protein in subcellular level changes, provide a good foundation, so as to discover and identify the protein and gene specific functions; third is to find and solve some problems such as biology, find new vaccine targets the place was also a pathogenic study for future vaccine development (screening, immune response antigen) provides an important reference for other related work. Therefore, we by the local epidemic strain screening Pick out the strong virulent strain CMCC50973. The expression of the protein. This experiment using pH4.0-5.0, pH4.5-5.5, pH5.0-6.0, alkaline narrow gradient strips and pH6-11 pH5.5-6.7, of CMCC50973 logarithmic growth stage of whole cell protein by two-dimensional electrophoresis, and then to test the protein spots visible to the horse Coomassie brilliant blue gum from the point of in gel digestion, identified by MALDI-TOF/TOF-MS. A total of 848 protein spots, 705 protein spots were identified, representing 519 genes encoding product, has a plurality of protein spots were identified as the same gene product as identified by the test protein. The calculated molecular weight and isoelectric that is consistent with predicted genome annotation values, but also there are some differences between the calculated value and the theoretical value. Some of the proteins identified in proteins is the most function and energy metabolism, nutrient transport and metabolism Related; mainly way in glycolysis, tricarboxylic acid cycle three, the pentose phosphate pathway, purine and pyrimidine metabolism and other metabolic pathways. 67 proteins in the test identified was annotated as hypothetical protein (hypotheticalprotein), the function is not clear for whole cell proteome map of.CMCC50973, to help us from the overall level of understanding of the genomic information for the study of protein expression, bacterial pathogenicity and host specificity, the expression of the genome, looking for new vaccine target, as well as providing a basis for immune response to antigen screening and other related work.
The outer membrane protein (outermembraneprotein, OMP), is the main structure of the outer membrane of gram negative bacteria, bacterial adhesion, invasion, etc. are produced in the process of drug resistance plays a very important role. Therefore, in the process of developing this kind of vaccine against pathogenic bacteria, study and screening of outer membrane protein, has become a very important of all kinds in the process of vaccine development. Our preliminary tests showed that the serum of Salmonella paratyphi A outer membrane protein BtuB and paratyphoid fever patients have a strong immune response, in order to further study the disease free protective proteins, we cloned in Escherichia coli, the expression of the protein after purification, purity was obtained. For the 90% rBtuB protein in mice showed that the recombinant protein has good immunogenicity. But the protection of attacking poison rate was only 60%, it can be used in combination with carrier vaccine, and other anti It was used in common use.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378

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