布鲁氏菌外膜蛋白免疫蛋白质组学的初步研究

发布时间：2018-08-16 13:16

【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。世界上170多个国家和地区有人、畜布病的存在和流行。在我国布病波及28个省市区,并且近年来死灰复燃,给畜牧业和人类的健康带来严重危害。布鲁氏菌包括新发现的海洋哺乳动物种在内,有7种21个生物型,感染人的主要有牛、羊、猪、犬和新发现的海洋种。布鲁氏菌属革兰氏阴性菌,兼性寄生于巨噬细胞内,从而逃避宿主的免疫清除。它主要引起人的波状热和慢性感染,造成反刍动物不育和流产等,同时又是一种潜在的生物战剂和生物恐怖剂。因布病独特的致病机制和感染后难以根治的特点,疫苗一直在布病的综合防治措施中占有极为重要的地位。其中减毒活疫苗应用较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问题。随着羊种、猪种和牛种布鲁氏菌基因组测序的完成和反向疫苗学理论和技术的发展,使布病疫苗研究进入“后基因组学”时代。“后基因组学”时代迫切需要解决现有疫苗的缺陷,即“安全性不足”和“保护效力有限”。目前研究表明布鲁氏菌重组亚单位疫苗能产生一定的免疫原性和免疫保护性,但是目前发现的分子保护效果不完全,提示还有新的保护性抗原没有被发现,因此找到布病疫苗新靶标是发展新一代疫苗的重大科学问题。基于以上思路,本研究对我国羊种布鲁氏菌五号菌种简称M5的外膜蛋白进行免疫蛋白质组学的初步研究,以期筛选布鲁氏菌保护性抗原分子,为安全、有效的布鲁氏菌重组亚单位疫苗研制提供保证。本研究的主要内容包括以下几方面: (1)制备布鲁氏菌免疫血清(兔血清),然后制备布鲁氏菌M5的外膜蛋白样品,通过双向电泳分离外膜蛋白质样品,将蛋白质转移到PVDF膜上,利用兔血清通过Western blot来探测免疫蛋白点。21个免疫蛋白点经胶内酶切、肽提取后用基质辅助激光解析/电子飞行时间质谱(MALDI-TOF MS)进行鉴定。每个蛋白点的肽质量指纹图谱用Mascot进行检索后,代表了12个开放阅读框。这些蛋白不全是外膜蛋白,还存在胞浆蛋白,其功能涉及生物合成和物质代谢等领域,还有一个功能未知的蛋白。结果成功建立了布鲁氏菌外膜蛋白的免疫蛋白质组学研究方法,不仅验证了传统使用的抗原,同时也鉴定了新的免疫分子。为寻找保护性抗原分子及新型疫苗抗原候选提供新思路。 (2)通过文献调研和生物信息学的分析,本部分选取5个免疫蛋白基因,进行PCR扩增目的片段,分别构建在原核表达载体pET32a上,转化大肠埃希菌BL21,经诱导表达、纯化相应的蛋白,结果成功的诱导表达了4个免疫蛋白并进行了Western blot的鉴定具有反应原性。其中一个未诱导成功,条件正在摸索中。同时将5个蛋白基因构建到真核表达载体pVAX1上,转染COS-7细胞后验证有目的蛋白表达,大提质粒制备相应的候选DNA疫苗,构建基因工程疫苗。免疫Balb/c小鼠,共免疫3次,前2次用重组基因产物免疫,后1次用相应重组蛋白产物加强免疫。通过ELISA、ELISPOT、FCM及MTT来检测疫苗在免疫小鼠体内诱导产生的体液及细胞免疫指标。结果表明DNA候选疫苗可同时激发体液免疫和细胞免疫。以上研究为基于疫苗策略的有效预防布病的深入研究奠定了基础。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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