靶向化布鲁氏菌树突状细胞疫苗的试制研究

发布时间：2018-01-11 14:25

本文关键词：靶向化布鲁氏菌树突状细胞疫苗的试制研究　出处：《河北农业大学》2011年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌病是由布鲁氏菌引起的一种人畜共患传染病。布鲁氏菌主要经由粘膜和伤口感染,能够引起家畜的自发性流产和人的“波浪热”。大量资料显示,布鲁氏菌的易感细胞是巨噬细胞,该细胞感染布鲁氏菌后,在其胞内大量繁殖,使其抗原提呈能力丧失,从而使宿主的免疫功能破坏而引发持续性感染。因此,如何帮助患病动物建立有效的免疫应答机制是当前研究的热点。树突状细胞(dendritic cell, DC)是迄今为止发现的体内功能最强的专职抗原提呈细胞。适应性免疫应答的启动需要DC的抗原提呈。DC不仅能刺激已活化或记忆性的T细胞,而且能显著刺激初始T细胞的增殖,它触发的是点放射状放大反应,1个DC可以激活100～3000个T细胞。因此DC是体内免疫反应的始动者,在免疫治疗中有着巨大的魅力。本实验围绕DC进行疫苗研究,并在动物试验中检验实验疫苗的免疫效果。研究表明,布鲁氏菌的外膜蛋白(Outer membrane proteins, OMPs)有很强的免疫原性。本实验采用去污剂连续抽提法提取了布鲁氏菌的全外膜蛋白,经SDS-PAGE电泳后,选择了抗原表位和糖基化位点较多的37.5 kD与47.2 kD的蛋白作为疫苗抗原候选蛋白,经过分离、电洗脱纯化得到这两种布鲁氏菌OMPs。采用α-D-甘露吡喃异硫氰酸苯酯对这两种蛋白质抗原进行甘露糖修饰,以靶向体内抗原提呈功能最强的专职抗原提呈细胞—树突状细胞,再经间苯二酚-硫酸法鉴定两种蛋白质抗原糖基修饰效果。采取方差分析比较布鲁氏菌外膜蛋白糖基修饰后的免疫效果,以血清中抗体水平与淋巴细胞增殖能力作为检测指标,随机选取20只小鼠,并随机分成A、B、C和D四组,分别肌注布鲁氏菌S2商品疫苗、纯化的布鲁氏菌两种混合外膜蛋白、甘露糖修饰化的布鲁氏菌外膜蛋白和PBS。试验结果表明:二免后4d、7d、10d ,A组、B组和C组间血清抗体水平差异不显著(P0.05),但A组、B组、C组与D组相比差异极显著(P0.01),具有统计学意义,表明布鲁氏菌S2商品疫苗、布鲁氏菌两种混合外膜蛋白、修饰化的布鲁氏菌外膜蛋白免疫小鼠后,血清抗体水平均显著高于阴性对照组,明显诱导了机体的体液免疫应答。由淋巴细胞增殖试验表明:C组内的实验组OD值/对照组OD值明显高于A组、B组、D组。说明小鼠免疫甘露糖修饰化的布鲁氏菌外膜蛋白在二免10d后,T淋巴细胞增殖能力明显高于免疫布鲁氏菌S2商品疫苗、纯化的布鲁氏菌两种混合外膜蛋白与PBS对照组。表明甘露糖修饰化的布鲁氏菌外膜蛋白在诱导机体的细胞免疫应答发挥了作用。以上实验结果证实蛋白质经糖基化修饰后有显著免疫效果。疫苗佐剂的选择直接影响着疫苗的免疫效果,为了使疫苗的效果达到最佳,选择了法国赛比克公司的四种佐剂Montanide ISA 71 VG、ESSAI ISA 206 CEL、Montanide IMS 251C VG和Montanide Gel 01 PR,利用糖基化布鲁氏菌OMPs制作了四种不同佐剂的疫苗。由正交试验筛选布鲁氏菌外膜蛋白的配合佐剂,最佳免疫程序。结果显示:抗原使用Montanide IMS 251C VG纳米颗粒佐剂,免疫剂量为甘露糖基化修饰的布鲁氏菌外膜蛋白X1μg,采取肌内注射,免疫三次这种组合,无论是在抗体产生趋势还是血清IFN-γ产生水平为最佳组合。完全随机试验、腹腔巨噬细胞吞噬试验进一步检验靶向化布鲁氏菌树突状细胞疫苗免疫效果,结果表明靶向化疫苗诱导了机体较强的免疫应答。安全性试验结果表明:靶向化布鲁氏菌外膜蛋白树突状细胞疫苗对6~8周龄BALB/c健康小鼠、妊娠小鼠均安全。本研究的实验结果为基于疫苗研究策略,有效预防布鲁氏病奠定了基础。
[Abstract]:Brucellosis caused by Brucella is a zoonotic infectious disease. Brucella mainly through the mucosa and wound infection, can cause livestock spontaneous abortion and "heat wave". A large number of data showed that Brucella susceptible cells are macrophages, the cells infected with Brucella, multiply in the intracellular, the antigen presenting capacity loss, so that the host immune function damage caused by persistent infection. Therefore, how to help the sick animal immune response effectively is the focus of current research.
Dendritic cells (dendritic, cell, DC) are the most potent antigen found so far presenting cells. Adaptive immune response needs to start the antigen presentation of DC.DC can not only stimulate the activated and memory T cells, and can significantly stimulate initial T cell proliferation, it is triggered by the radial point the amplification reaction, 1 DC can activate 100 ~ 3000 T cells. Therefore DC is the initiator of immune response in the body, has great charm in immunotherapy. The experimental vaccine research on DC, and test the immune effect of vaccine in animal experiment.
Research shows that the Brucella outer membrane protein (Outer membrane, proteins, OMPs) have strong immunogenicity. This experiment used detergent continuous extraction the Brucella outer membrane protein, by SDS-PAGE electrophoresis, the epitope and glycosylation sites are 37.5 kD and 47.2 kD protein as antigen vaccine candidate protein, after separation, electroelution of these two kinds of Brucella OMPs. by alpha -D- mannan pyran phenyl isothiocyanate mannose modification of these two protein antigens, in vivo targeting antigen presenting the most potent antigen presenting cells, dendritic cells, and then through the identification of two kinds of resorcinol sulfuric acid method protein glycosylation effect.
Take the immune effect of variance analysis of Brucella outer membrane protein glycosylation after modification, with antibody level and lymphocyte proliferation in serum as indexes, 20 mice were randomly selected, and were randomly divided into A, B, C and D four groups, respectively with S2 of Brucella vaccine two kinds of mixed goods, purification of the Brucella outer membrane protein of Brucella. The outer membrane protein and PBS. test results show that the mannose modified after two free 4D, 7d, 10d, A group, serum antibody levels between B group and C group were not significant (P0.05), A group, B group, C group and D group compared the difference was statistically significant (P0.01). S2, show that the two kinds of commodity Brucella vaccine, hybrid membrane protein of Brucella, modified Brucella outer membrane protein of mice, serum antibody levels were significantly higher than the negative control group, significantly induced humoral immune responses by lymphocytes increased. Cloning experiments showed that the OD value of experimental group / group C in control group was significantly higher than that of A group, B group, D group. The mice immune mannose modified Brucella outer membrane protein in two free 10d, T lymphocyte proliferation was significantly higher than that of immune Brucella S2 commodity vaccine, cloth Lu's purified two hybrid membrane protein and PBS control group. Showed that mannose modification of Brucella outer membrane protein play a role in the cellular immune response induced by the body. The above results confirmed that the protein was glycosylated with significant immune effect.
Vaccine adjuvants directly affect the immune effects of the vaccine, the vaccine in order to achieve the best results, choose four kinds of adjuvant Montanide ISA SEPPIC 71 VG, 206 ESSAI ISA CEL Montanide IMS, 251C VG and Montanide Gel 01 PR, the glycosylation of Brucella strains OMPs were produced in four different adjuvants with the adjuvant vaccine. By orthogonal test of Brucella outer membrane protein, the best immune procedure. The results showed that the use of Montanide IMS 251C antigen VG nanoparticle adjuvant, immune dose of mannosylation modified Brucella outer membrane protein X1 G by intramuscular injection, immune three times this combination, whether in the trend or serum antibody production IFN- gamma production level is the best combination.
Completely random test, peritoneal macrophage phagocytosis test to further examine the targeting effect of DCS vaccine immune. The results show that the target induced immune response to the vaccine. The strong safety test results show that the targeted vaccine of Brucella outer membrane protein dendritic cells to 6~8 week old BALB/c mice, pregnant mice were safe.
The results of this research for vaccine research strategy based on the effective prevention of brucellosis has laid the foundation.

【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【引证文献】