本文關(guān)鍵詞：布魯氏菌毒力、免疫相關(guān)分子的篩選研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 布魯氏菌 蛋白質(zhì)組學(xué) 反向疫苗學(xué) 新型疫苗 雙向電泳

【摘要】： 布魯氏菌病(Brucellosis)(簡(jiǎn)稱布病)是由布魯氏菌(Brucella)引起的人畜共患傳染病,在我國被列為二類傳染病,是一種重要的生物戰(zhàn)劑和生物恐怖劑。近年來,疫情反彈,死灰復(fù)燃,給畜牧業(yè)和人類健康帶來嚴(yán)重危害。布魯氏菌屬有7個(gè)種,感染人的主要有牛、羊、豬、犬及海洋動(dòng)物5個(gè)種。布魯氏菌是革蘭氏陰性菌,主要引起人的波狀熱和慢性感染以及反芻動(dòng)物的不育和流產(chǎn)。 疫苗是防控布病最有效、最經(jīng)濟(jì)的手段。其中畜用減毒活疫苗應(yīng)用較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問題。至今還沒有一種安全、有效的人用疫苗。人們對(duì)布魯氏菌的致病機(jī)理和宿主的免疫保護(hù)機(jī)制等方面認(rèn)識(shí)還相當(dāng)膚淺,限制了新型疫苗的研制進(jìn)度。 羊種、豬種和牛種布魯氏菌基因組測(cè)序和注釋完成使布病研究進(jìn)入“后(功能)基因組學(xué)”時(shí)代�！昂蠡蚪M學(xué)”時(shí)代迫切需要解決現(xiàn)有疫苗的兩大缺陷,即“安全性不足”和“保護(hù)效力有限”。闡明布魯氏菌毒力、免疫相關(guān)分子無疑成為解決現(xiàn)有疫苗缺陷、研制新型疫苗的突破口。隨著“后基因組學(xué)”研究的深入,反向疫苗學(xué)應(yīng)運(yùn)而生,為新型疫苗的研制提供了理論和技術(shù)支持。 本研究通過蛋白質(zhì)組學(xué)初步挖掘布魯氏菌毒力、免疫相關(guān)分子,通過分子生物學(xué)和疫苗學(xué)初步驗(yàn)證其免疫功能,以深化人們對(duì)布魯氏菌毒力和免疫機(jī)制的認(rèn)識(shí),打破布病新型疫苗研制的瓶頸,為布病防治提供新的依據(jù)。 首先,成功提取布魯氏菌的全細(xì)菌蛋白和外膜蛋白,并分別進(jìn)行了2-DE,獲得了很好的蛋白分離效果,為布魯氏菌蛋白質(zhì)組學(xué)研究奠定了良好的基礎(chǔ)。 其次,成功地對(duì)我國現(xiàn)用布病弱毒疫苗株B.melitensis M5和國際標(biāo)準(zhǔn)強(qiáng)毒株B.melitensis 16M進(jìn)行了初步的比較蛋白質(zhì)組學(xué)研究。在4pI7,10kDMW120kD的2-DE窗口上,共發(fā)現(xiàn)14個(gè)差別表達(dá)的蛋白質(zhì)點(diǎn),通過質(zhì)譜分析并進(jìn)行Mascot檢索,獲得了8個(gè)ORF。這些蛋白涉及代謝相關(guān)的酶、分子伴侶等,通過分析表明M5的致弱機(jī)制與美國現(xiàn)用布病弱毒疫苗株B.melitensis Rev 1明顯不同,為進(jìn)一步深入研究布魯氏菌的毒力機(jī)制奠定了基礎(chǔ)。 再次,成功地對(duì)M5的全細(xì)菌蛋白和外膜蛋白用布魯氏菌免疫兔陽性血清進(jìn)行了免疫蛋白質(zhì)組學(xué)研究。在4pI7,10kDMW120kD的2-DE窗口上,在外膜蛋白中,共發(fā)現(xiàn)21個(gè)具有免疫原性的蛋白質(zhì)點(diǎn),通過質(zhì)譜分析并進(jìn)行Mascot檢索,獲得了12個(gè)ORF。在全細(xì)菌蛋白中,共發(fā)現(xiàn)67個(gè)具有免疫原性的蛋白質(zhì)點(diǎn),Mascot檢索后,獲得了57個(gè)ORF。與此同時(shí),用病人、病牛、病羊等臨床陽性血清進(jìn)行了初步篩選,進(jìn)行了初步的條件優(yōu)化,獲得了一批免疫蛋白分子。為布魯氏菌的檢測(cè)、診斷及防治藥物靶點(diǎn)提供了新的依據(jù),為疫苗候選新抗原篩選奠定了理論基礎(chǔ)。 最后,優(yōu)選5個(gè)基因進(jìn)行了基因的克隆、表達(dá)、純化及免疫小鼠進(jìn)行了免疫保護(hù)性相關(guān)評(píng)價(jià)。通過WB、ELISA、ELISPOT、MTT、FCM等免疫學(xué)檢測(cè),初步表明5個(gè)蛋白均有免疫性,3個(gè)蛋白可能具有保護(hù)性。為高通量、大規(guī)模地表達(dá)、純化布魯氏菌蛋白,進(jìn)一步研究其功能奠定了技術(shù)基礎(chǔ),同時(shí)為大規(guī)模評(píng)價(jià)抗原的免疫保護(hù)性奠定了技術(shù)基礎(chǔ)。 通過本研究,獲得了以下結(jié)果和認(rèn)識(shí): 1. Rev1和M5雖同為Ⅰ型羊種布魯氏菌的疫苗株,其致弱分子機(jī)制卻不同。暗示對(duì)傳統(tǒng)疫苗株致弱分子機(jī)制進(jìn)行深入研究有重要的價(jià)值。同時(shí)發(fā)現(xiàn)BMEⅡ0590編碼的Sugar-binding protein和BMEⅠ1980編碼的Dps在16M、M5、Rev1表達(dá)差異顯著,有重要的研究?jī)r(jià)值,有必要采用分子遺傳學(xué)方法進(jìn)行深入研究。 2.通過多種宿主陽性血清篩選多種布魯氏菌不同組分能夠相互補(bǔ)充,最大限度發(fā)掘抗原。但僅借助蛋白質(zhì)組學(xué)技術(shù)仍然無法發(fā)掘所有的抗原,需借助生物信息學(xué)、生物芯片等技術(shù)進(jìn)行深入發(fā)掘。 3.篩選到60多個(gè)抗原分子,其中10多個(gè)在其它研究中得到確認(rèn),其余的為新發(fā)現(xiàn)的抗原分子,急需研究其免疫性和保護(hù)性。 4.克隆、表達(dá)、純化了2個(gè)全長(zhǎng)外膜蛋白分子和3個(gè)外膜蛋白分子片斷,為大規(guī)模表達(dá)積累了經(jīng)驗(yàn)。對(duì)5個(gè)分子進(jìn)行免疫學(xué)評(píng)價(jià),初步表明BMEⅠ1007、BMEⅡ0105、BMEⅠ1069具有保護(hù)潛能。 以上研究為成功運(yùn)用反向疫苗學(xué)理論和技術(shù)研制布病新型疫苗提供了蛋白質(zhì)組學(xué)和免疫學(xué)方面技術(shù)平臺(tái)。
[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，

本文編號(hào)：1381724