【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(guó)被列為二類傳染病。世界上170多個(gè)國(guó)家和地區(qū)有人、畜布病的存在和流行。在我國(guó)布病波及28個(gè)省市區(qū),并且近年來(lái)死灰復(fù)燃,給畜牧業(yè)和人類的健康帶來(lái)嚴(yán)重危害。 布魯氏菌包括新發(fā)現(xiàn)的海洋哺乳動(dòng)物種在內(nèi),有7種21個(gè)生物型,感染人的主要有牛、羊、豬、犬和新發(fā)現(xiàn)的海洋種。布魯氏菌屬革蘭氏陰性菌,兼性寄生于巨噬細(xì)胞內(nèi),從而逃避宿主的免疫清除。它主要引起人的波狀熱和慢性感染,造成反芻動(dòng)物不育和流產(chǎn)等,同時(shí)又是一種潛在的生物戰(zhàn)劑和生物恐怖劑。 因布病獨(dú)特的致病機(jī)制和感染后難以根治的特點(diǎn),疫苗一直在布病的綜合防治措施中占有極為重要的地位。其中減毒活疫苗應(yīng)用較早至今仍占主要地位,但在安全性和有效性方面仍存在很多問題。隨著羊種、豬種和牛種布魯氏菌基因組測(cè)序的完成和反向疫苗學(xué)理論和技術(shù)的發(fā)展,使布病疫苗研究進(jìn)入“后基因組學(xué)”時(shí)代。“后基因組學(xué)”時(shí)代迫切需要解決現(xiàn)有疫苗的缺陷,即“安全性不足”和“保護(hù)效力有限”。目前研究表明布魯氏菌重組亞單位疫苗能產(chǎn)生一定的免疫原性和免疫保護(hù)性,但是目前發(fā)現(xiàn)的分子保護(hù)效果不完全,提示還有新的保護(hù)性抗原沒有被發(fā)現(xiàn),因此找到布病疫苗新靶標(biāo)是發(fā)展新一代疫苗的重大科學(xué)問題�；谝陨纤悸�,本研究對(duì)我國(guó)羊種布魯氏菌五號(hào)菌種簡(jiǎn)稱M5的外膜蛋白進(jìn)行免疫蛋白質(zhì)組學(xué)的初步研究,以期篩選布魯氏菌保護(hù)性抗原分子,為安全、有效的布魯氏菌重組亞單位疫苗研制提供保證。本研究的主要內(nèi)容包括以下幾方面: (1)制備布魯氏菌免疫血清(兔血清),然后制備布魯氏菌M5的外膜蛋白樣品,通過(guò)雙向電泳分離外膜蛋白質(zhì)樣品,將蛋白質(zhì)轉(zhuǎn)移到PVDF膜上,利用兔血清通過(guò)Western blot來(lái)探測(cè)免疫蛋白點(diǎn)。21個(gè)免疫蛋白點(diǎn)經(jīng)膠內(nèi)酶切、肽提取后用基質(zhì)輔助激光解析/電子飛行時(shí)間質(zhì)譜(MALDI-TOF MS)進(jìn)行鑒定。每個(gè)蛋白點(diǎn)的肽質(zhì)量指紋圖譜用Mascot進(jìn)行檢索后,代表了12個(gè)開放閱讀框。這些蛋白不全是外膜蛋白,還存在胞漿蛋白,其功能涉及生物合成和物質(zhì)代謝等領(lǐng)域,還有一個(gè)功能未知的蛋白。結(jié)果成功建立了布魯氏菌外膜蛋白的免疫蛋白質(zhì)組學(xué)研究方法,不僅驗(yàn)證了傳統(tǒng)使用的抗原,同時(shí)也鑒定了新的免疫分子。為尋找保護(hù)性抗原分子及新型疫苗抗原候選提供新思路。 (2)通過(guò)文獻(xiàn)調(diào)研和生物信息學(xué)的分析,本部分選取5個(gè)免疫蛋白基因,進(jìn)行PCR擴(kuò)增目的片段,分別構(gòu)建在原核表達(dá)載體pET32a上,轉(zhuǎn)化大腸埃希菌BL21,經(jīng)誘導(dǎo)表達(dá)、純化相應(yīng)的蛋白,結(jié)果成功的誘導(dǎo)表達(dá)了4個(gè)免疫蛋白并進(jìn)行了Western blot的鑒定具有反應(yīng)原性。其中一個(gè)未誘導(dǎo)成功,條件正在摸索中。同時(shí)將5個(gè)蛋白基因構(gòu)建到真核表達(dá)載體pVAX1上,轉(zhuǎn)染COS-7細(xì)胞后驗(yàn)證有目的蛋白表達(dá),大提質(zhì)粒制備相應(yīng)的候選DNA疫苗,構(gòu)建基因工程疫苗。免疫Balb/c小鼠,共免疫3次,前2次用重組基因產(chǎn)物免疫,后1次用相應(yīng)重組蛋白產(chǎn)物加強(qiáng)免疫。通過(guò)ELISA、ELISPOT、FCM及MTT來(lái)檢測(cè)疫苗在免疫小鼠體內(nèi)誘導(dǎo)產(chǎn)生的體液及細(xì)胞免疫指標(biāo)。結(jié)果表明DNA候選疫苗可同時(shí)激發(fā)體液免疫和細(xì)胞免疫。以上研究為基于疫苗策略的有效預(yù)防布病的深入研究奠定了基礎(chǔ)。
[Abstract]:Brucellosis (Brucellosis) is an infectious disease caused by Brucella. It is classified as the second kind of infectious disease in China. Brucellosis exists and prevails in more than 170 countries and regions in the world. Brucellosis has spread to 28 provinces and municipalities in China. In recent years, brucellosis has been resurgence, which has brought about a serious threat to animal husbandry and human health. Bring serious harm.
Brucella, including newly discovered marine mammal species, has 7 species and 21 biological types. It infects mainly human beings * cattle, sheep, pigs, dogs and newly discovered marine species. Brucella belongs to Gram-negative bacteria, facultative parasitic macrophages, thus evading host immune clearance. It mainly causes the wave fever and chronic infection of human beings, causing ruminants. Animal sterility and abortion are also potential biological agents and bioterrorism agents.
Because of the unique pathogenic mechanism of brucellosis and the difficulty in eradication of infection, vaccines have been playing a very important role in the comprehensive prevention and treatment of brucellosis. The application of live attenuated vaccines is still dominant, but there are still many problems in terms of safety and efficacy. * With the completion of sequencing and the development of reverse vaccines theory and technology, the study of brucellosis vaccine has entered the era of "post-genomics". Immunogenicity and immune protection, but the molecular protective effect of Brucella spp. 5 is not complete, suggesting that there are still new protective antigens not found, so finding a new target of Brucella spp. vaccine is a major scientific issue for the development of a new generation of vaccines. The aim of this study is to screen the protective antigens of Brucella spp. and to provide a guarantee for the development of a safe and effective recombinant Brucella subunit vaccine.
(1) Brucella immune serum (rabbit serum) was prepared, then Brucella M5 outer membrane protein samples were prepared. The outer membrane protein samples were separated by two-dimensional electrophoresis and transferred to PVDF membrane. The immune protein spots were detected by Western blot. 21 immune protein spots were digested by in-gel enzyme digestion and extracted by matrix-assisted laser. Analytical/Electronic Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was used to identify the peptides. The peptide mass fingerprints of each protein point were retrieved by Mascot and represented 12 open reading frames. These proteins were not only outer membrane proteins, but also cytoplasmic proteins. Their functions were involved in biosynthesis and material metabolism, and there was a protein with unknown function. The Immunoproteomics method of Brucella outer membrane proteins was successfully established, which not only verified the antigens used traditionally, but also identified new immune molecules.
(2) Through literature research and bioinformatics analysis, five immune protein genes were selected and amplified by PCR. The target fragments were constructed on prokaryotic expression vector pET32a and transformed into Escherichia coli BL21. The corresponding proteins were induced to express and purified. The results showed that four immune proteins were successfully induced and expressed and identified by Western blot. One of them was not induced successfully, and the conditions were being explored. At the same time, five protein genes were constructed on the eukaryotic expression vector pVAX1. After transfection into COS-7 cells, the expression of the target protein was verified. The corresponding candidate DNA vaccine was prepared by large-scale plasmid extraction, and a genetic engineering vaccine was constructed. The humoral and cellular immune indices induced by the vaccine were detected by ELISA, ELISPOT, FCM and MTT. The results showed that DNA vaccine candidates could stimulate both humoral and cellular immunity. It laid a foundation for further research.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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