布鲁氏菌毒力、免疫相关分子的筛选研究

发布时间：2018-01-05 05:18

  本文关键词：布鲁氏菌毒力、免疫相关分子的筛选研究　出处：《中国人民解放军军事医学科学院》2008年博士论文　论文类型：学位论文

  更多相关文章： 布鲁氏菌 蛋白质组学 反向疫苗学 新型疫苗 双向电泳

【摘要】： 布鲁氏菌病(Brucellosis)(简称布病)是由布鲁氏菌(Brucella)引起的人畜共患传染病,在我国被列为二类传染病,是一种重要的生物战剂和生物恐怖剂。近年来,疫情反弹,死灰复燃,给畜牧业和人类健康带来严重危害。布鲁氏菌属有7个种,感染人的主要有牛、羊、猪、犬及海洋动物5个种。布鲁氏菌是革兰氏阴性菌,主要引起人的波状热和慢性感染以及反刍动物的不育和流产。 疫苗是防控布病最有效、最经济的手段。其中畜用减毒活疫苗应用较早且至今仍占主要地位,但在安全性和有效性方面存在很多问题。至今还没有一种安全、有效的人用疫苗。人们对布鲁氏菌的致病机理和宿主的免疫保护机制等方面认识还相当肤浅,限制了新型疫苗的研制进度。 羊种、猪种和牛种布鲁氏菌基因组测序和注释完成使布病研究进入“后(功能)基因组学”时代。“后基因组学”时代迫切需要解决现有疫苗的两大缺陷,即“安全性不足”和“保护效力有限”。阐明布鲁氏菌毒力、免疫相关分子无疑成为解决现有疫苗缺陷、研制新型疫苗的突破口。随着“后基因组学”研究的深入,反向疫苗学应运而生,为新型疫苗的研制提供了理论和技术支持。 本研究通过蛋白质组学初步挖掘布鲁氏菌毒力、免疫相关分子,通过分子生物学和疫苗学初步验证其免疫功能,以深化人们对布鲁氏菌毒力和免疫机制的认识,打破布病新型疫苗研制的瓶颈,为布病防治提供新的依据。 首先,成功提取布鲁氏菌的全细菌蛋白和外膜蛋白,并分别进行了2-DE,获得了很好的蛋白分离效果,为布鲁氏菌蛋白质组学研究奠定了良好的基础。 其次,成功地对我国现用布病弱毒疫苗株B.melitensis M5和国际标准强毒株B.melitensis 16M进行了初步的比较蛋白质组学研究。在4pI7,10kDMW120kD的2-DE窗口上,共发现14个差别表达的蛋白质点,通过质谱分析并进行Mascot检索,获得了8个ORF。这些蛋白涉及代谢相关的酶、分子伴侣等,通过分析表明M5的致弱机制与美国现用布病弱毒疫苗株B.melitensis Rev 1明显不同,为进一步深入研究布鲁氏菌的毒力机制奠定了基础。 再次,成功地对M5的全细菌蛋白和外膜蛋白用布鲁氏菌免疫兔阳性血清进行了免疫蛋白质组学研究。在4pI7,10kDMW120kD的2-DE窗口上,在外膜蛋白中,共发现21个具有免疫原性的蛋白质点,通过质谱分析并进行Mascot检索,获得了12个ORF。在全细菌蛋白中,共发现67个具有免疫原性的蛋白质点,Mascot检索后,获得了57个ORF。与此同时,用病人、病牛、病羊等临床阳性血清进行了初步筛选,进行了初步的条件优化,获得了一批免疫蛋白分子。为布鲁氏菌的检测、诊断及防治药物靶点提供了新的依据,为疫苗候选新抗原筛选奠定了理论基础。 最后,优选5个基因进行了基因的克隆、表达、纯化及免疫小鼠进行了免疫保护性相关评价。通过WB、ELISA、ELISPOT、MTT、FCM等免疫学检测,初步表明5个蛋白均有免疫性,3个蛋白可能具有保护性。为高通量、大规模地表达、纯化布鲁氏菌蛋白,进一步研究其功能奠定了技术基础,同时为大规模评价抗原的免疫保护性奠定了技术基础。 通过本研究,获得了以下结果和认识: 1. Rev1和M5虽同为Ⅰ型羊种布鲁氏菌的疫苗株,其致弱分子机制却不同。暗示对传统疫苗株致弱分子机制进行深入研究有重要的价值。同时发现BMEⅡ0590编码的Sugar-binding protein和BMEⅠ1980编码的Dps在16M、M5、Rev1表达差异显著,有重要的研究价值,有必要采用分子遗传学方法进行深入研究。 2.通过多种宿主阳性血清筛选多种布鲁氏菌不同组分能够相互补充,最大限度发掘抗原。但仅借助蛋白质组学技术仍然无法发掘所有的抗原,需借助生物信息学、生物芯片等技术进行深入发掘。 3.筛选到60多个抗原分子,其中10多个在其它研究中得到确认,其余的为新发现的抗原分子,急需研究其免疫性和保护性。 4.克隆、表达、纯化了2个全长外膜蛋白分子和3个外膜蛋白分子片断,为大规模表达积累了经验。对5个分子进行免疫学评价,初步表明BMEⅠ1007、BMEⅡ0105、BMEⅠ1069具有保护潜能。 以上研究为成功运用反向疫苗学理论和技术研制布病新型疫苗提供了蛋白质组学和免疫学方面技术平台。
[Abstract]:brucella is a kind of infectious disease caused by brucella , which is classified as Class II infectious disease in our country . It is an important biological warfare agent and bioterrorism agent . In recent years , the epidemic has rebounded , the resurgence of death has caused serious harm to animal husbandry and human health . The brucella is Gram - negative bacteria , which is mainly responsible for the wave fever and chronic infection of human and the infertility and abortion of ruminant . The vaccine is the most effective and economical means for preventing and controlling the disease . Many problems exist in the application of attenuated live vaccine for livestock , but there are many problems in safety and effectiveness . The genome sequencing and annotation of melitensis , pig and cattle have completed the " post - functional genomics " era . The " post - genomics " era urgently needs to solve the two major defects of the existing vaccine , namely " insufficient safety " and " limited protection potency " . It is no doubt that the virulence of brucella is " insufficient safety " and " limited protection potency " . As the research of " post - genomics " is in - depth , the reverse vaccine is born , which provides theoretical and technical support for the development of new vaccines . In this study , we preliminarily verified the virulence and immune - related molecules of brucella by Proteomics , and preliminarily verified its immune function through molecular biology and vaccine , so as to deepen the understanding of the virulence and immune mechanism of brucella , and break the bottleneck of new vaccine development , and provide a new basis for the prevention and control of the disease . Firstly , the whole bacterial proteins and outer membrane proteins of brucella were successfully extracted , and 2 - DE was obtained . A preliminary comparative proteomic study was carried out on B . melitensis M5 and B . melitensis 16M . In the 4pI7,10kDMW120kD 2 - DE window , 14 differentially expressed protein spots were found , and 8 ORFs were obtained by mass spectrometry and Mascot search . These proteins were involved in metabolism - related enzymes , molecular couples , etc . The analysis showed that the weak mechanism of M5 was different from that of B . melitensis Rev 1 in the United States . At the same time , there were 67 immunogenic protein spots in the 2 - DE window of 4pI7 and 10kDMW120kD . Finally , 5 genes were cloned , expressed , purified and immunized with immune protective immunity . By means of the immunological detection of WB , ELISA , POT , MTT , FCM and so on , it was shown that 5 proteins were immune . Three proteins could be protective . For high - throughput , large - scale expression and purification of brucellin , the functional foundation was established , and the technical foundation was established for the immune protection of large - scale evaluation antigen . Through this study , the following results and understandings are obtained : 1 . Although both Rev1 and M5 have different molecular mechanisms , it is very important to study the weak molecular mechanism of traditional vaccine strain . It is found that the Dps encoded by BME 鈪，

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