本文關(guān)鍵詞： 甲型副傷寒沙門菌 基因組學(xué) 蛋白質(zhì)組學(xué) 疫苗 外膜蛋白 BtuB蛋白　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文　論文類型：學(xué)位論文

【摘要】：甲型副傷寒沙門菌（SalmonellaParatyphiA，SPA）是甲型副傷寒（paratyphoidfeverA）的病原菌，通過糞－口途徑傳播，感染劑量為103～109個(gè)菌，能夠引起傷寒樣臨床癥狀。人是唯一宿主。人群對甲型副傷寒沙門菌普遍易感，但兒童和青壯年發(fā)病最高。近些年來，在世界范圍內(nèi)，傷寒發(fā)病率有所降低，而甲型副傷寒發(fā)病率有明顯升高。在我國，甲型副傷寒的發(fā)病率也相對較高，并在局部地區(qū)有爆發(fā)或流行。目前，舊的甲型副傷寒滅活疫苗由于副反應(yīng)太大，已停止使用，尚無新的有效疫苗預(yù)防甲型副傷寒。因此對甲型副傷寒沙門菌的研究也越來越引起腸道致病菌領(lǐng)域?qū)W者的重視。 當(dāng)前分子生物學(xué)、基因組學(xué)、蛋白組學(xué)、生物信息學(xué)等快速發(fā)展的學(xué)科為疫苗的研發(fā)提供了革命性手段。通過對基因組信息的分析，篩選候選蛋白質(zhì)抗原的方法被稱為反向疫苗學(xué)（reversevaccinology），包括以下主要步驟：在獲取病原體的基因組序列后，芯片分析預(yù)測編碼分泌蛋白、表面相關(guān)抗原和毒力因子；蛋白質(zhì)組學(xué)分析膜相關(guān)的蛋白；DNA微陣列鑒定高度表達(dá)的基因和體內(nèi)上調(diào)的基因；從中綜合分析可能的蛋白質(zhì)抗原，將它們高通量克隆并表達(dá)；進(jìn)行體內(nèi)或體外免疫學(xué)實(shí)驗(yàn)分析，篩選有效的候選疫苗。可見，基因組序列和生物信息學(xué)分析的快速發(fā)展使基因注釋不再是限定了總蛋白數(shù)和根據(jù)預(yù)測的功能分成幾個(gè)組，而是預(yù)測細(xì)菌的可能全部蛋白在細(xì)胞中定位和功能等的信息。此外，蛋白質(zhì)組學(xué)的發(fā)展也對疫苗研究起著積極的促進(jìn)作用。蛋白質(zhì)組學(xué)技術(shù)是對基因組學(xué)技術(shù)的補(bǔ)充，也是疫苗發(fā)展的有力技術(shù)支撐。綜合基因組學(xué)、生物信息學(xué)、DNA微陣列和蛋白質(zhì)組學(xué)技術(shù)，可以對存在于病原體的不同成分給予充分詳細(xì)的定性、定量測定，對研發(fā)亞單位疫苗有很大的幫助。 本研究所使用的甲型副傷寒沙門菌CMCC50973，是一株經(jīng)篩選的毒力較強(qiáng)的地方流行株，計(jì)劃用于甲型副傷寒亞單位疫苗的研究。為了全面了解其遺傳信息，我們提取了細(xì)菌的基因組DNA，委托深圳華大公司測定了CMCC50973的全基因組序列。最終得到1個(gè)Scaffold環(huán)，全長為4,608,196bp，GC%為52.24%。預(yù)測基因4618個(gè)，tRNA100種，rRNA7種,sRNA56種。利用KEGG、COG、SwissProt、TrEMBL和NR數(shù)據(jù)庫為基因進(jìn)行了注釋。CMCC50973的基因組通過與已完成測序的甲型副傷寒沙門菌ATCC9150的參考序列NC_006511進(jìn)行比較，一共找到了143個(gè)SNP位點(diǎn)，其中：在基因區(qū)的有112個(gè)；在基因間區(qū)的有31個(gè)，共找到12個(gè)InDel位點(diǎn)，其中插入位點(diǎn)6個(gè)，刪除位點(diǎn)6個(gè)。該菌全基因組序列的獲得，為進(jìn)一步探尋基因間相互作用、新的調(diào)控因子等微生物更詳盡的遺傳學(xué)和生物學(xué)信息、預(yù)測和篩選新的更特異的保護(hù)性抗原基因，并在此基礎(chǔ)上發(fā)展高效疫苗或經(jīng)過遺傳學(xué)操作改造疫苗菌株、構(gòu)建活疫苗以及發(fā)展基因工程菌載體等打下了基礎(chǔ)。 表達(dá)蛋白質(zhì)組學(xué)的研究是蛋白質(zhì)組學(xué)研究的基礎(chǔ)內(nèi)容。建立起細(xì)胞在正常生理?xiàng)l件下的蛋白質(zhì)參考圖譜和數(shù)據(jù)庫，首先能對細(xì)菌的蛋白質(zhì)表達(dá)規(guī)律有整體的認(rèn)識，為細(xì)菌的研究提供確切、寶貴的數(shù)據(jù)資料；其次能為后續(xù)的功能比較研究，如比較分析在變化了的條件下，蛋白質(zhì)表達(dá)量的變化、翻譯后的加工修飾、蛋白質(zhì)在亞細(xì)胞水平上的改變等，提供很好的基礎(chǔ)，從而發(fā)現(xiàn)和鑒定出特定功能的蛋白及其基因；第三是可以發(fā)現(xiàn)和解決一些生物學(xué)問題，如找到新的疫苗靶位，也為將來進(jìn)行病原性研究、疫苗研制（免疫反應(yīng)性抗原篩選）等相關(guān)工作提供重要的參考。因此，我們由地方流行株中篩選出來的毒力較強(qiáng)的菌株CMCC50973，進(jìn)行了全菌蛋白表達(dá)譜的研究。本試驗(yàn)利用pH4.0-5.0、pH4.5-5.5、pH5.0-6.0、pH5.5-6.7的窄梯度膠條和pH6-11的堿性膠條，對CMCC50973對數(shù)生長末期的全菌體蛋白進(jìn)行了雙向電泳分離，然后對考馬斯亮蘭膠上可見的蛋白點(diǎn)取點(diǎn)進(jìn)行膠內(nèi)酶切，用MALDI-TOF/TOF-MS鑒定。共取蛋白點(diǎn)848個(gè)，鑒定到705個(gè)蛋白質(zhì)點(diǎn)，代表519個(gè)基因編碼產(chǎn)物，有多個(gè)蛋白點(diǎn)鑒定為同一基因產(chǎn)物的現(xiàn)象。所鑒定蛋白通過試驗(yàn)計(jì)算得出的分子量和等電點(diǎn)，與基因組注釋中的預(yù)測值比較一致，但也有一些蛋白質(zhì)理論值與計(jì)算值間存在一些差異。所鑒定蛋白質(zhì)中多數(shù)功能是和能量代謝、營養(yǎng)素的運(yùn)輸和代謝相關(guān)；主要出現(xiàn)在糖酵解途徑、三羧酸循環(huán)、磷酸戊糖途徑、嘌呤和嘧啶的代謝等代謝通路中。在試驗(yàn)中鑒定到的67個(gè)蛋白被注釋為“假想蛋白”（hypotheticalprotein），功能尚不明確。CMCC50973的全菌蛋白質(zhì)組圖譜的獲得，，幫助我們從整體層面了解了該菌基因組的蛋白表達(dá)信息，為研究細(xì)菌的致病機(jī)理和宿主特異性、基因組的表達(dá)情況、尋找新的疫苗靶位、以及免疫反應(yīng)性抗原篩選等相關(guān)工作提供了基礎(chǔ)。 外膜蛋白（outermembraneprotein,OMP），是革蘭氏陰性菌外膜的主要組成結(jié)構(gòu)，在細(xì)菌的黏附、侵襲、耐藥性產(chǎn)生等過程中都有著非常重要的作用。因此，在開發(fā)該類病原菌的疫苗過程中，對外膜蛋白的研究和篩選，已經(jīng)成為各類疫苗研發(fā)過程中非常重要的內(nèi)容。我們前期的試驗(yàn)證明甲型副傷寒沙門菌外膜蛋白BtuB與甲型副傷寒恢復(fù)期病人的血清有強(qiáng)烈的免疫反應(yīng)，為了進(jìn)一步研究該蛋白的免疫保護(hù)性，我們在大腸桿菌中克隆、表達(dá)了該蛋白，經(jīng)純化后獲得了純度約為90%的rBtuB蛋白，小鼠免疫顯示重組蛋白具有良好的免疫原性。但對攻毒的保護(hù)率僅為60%，可考慮將其用于結(jié)合疫苗的載體，與其它抗原共同使用。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R378

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