發(fā)布時間：2018-01-11 14:25

  本文關鍵詞：靶向化布魯氏菌樹突狀細胞疫苗的試制研究　出處：《河北農(nóng)業(yè)大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 布魯氏菌 樹突狀細胞 甘露糖受體 靶向化

【摘要】：布魯氏菌病是由布魯氏菌引起的一種人畜共患傳染病。布魯氏菌主要經(jīng)由粘膜和傷口感染,能夠引起家畜的自發(fā)性流產(chǎn)和人的“波浪熱”。大量資料顯示,布魯氏菌的易感細胞是巨噬細胞,該細胞感染布魯氏菌后,在其胞內大量繁殖,使其抗原提呈能力喪失,從而使宿主的免疫功能破壞而引發(fā)持續(xù)性感染。因此,如何幫助患病動物建立有效的免疫應答機制是當前研究的熱點。 樹突狀細胞(dendritic cell, DC)是迄今為止發(fā)現(xiàn)的體內功能最強的專職抗原提呈細胞。適應性免疫應答的啟動需要DC的抗原提呈。DC不僅能刺激已活化或記憶性的T細胞,而且能顯著刺激初始T細胞的增殖,它觸發(fā)的是點放射狀放大反應,1個DC可以激活100～3000個T細胞。因此DC是體內免疫反應的始動者,在免疫治療中有著巨大的魅力。本實驗圍繞DC進行疫苗研究,并在動物試驗中檢驗實驗疫苗的免疫效果。 研究表明,布魯氏菌的外膜蛋白(Outer membrane proteins, OMPs)有很強的免疫原性。本實驗采用去污劑連續(xù)抽提法提取了布魯氏菌的全外膜蛋白,經(jīng)SDS-PAGE電泳后,選擇了抗原表位和糖基化位點較多的37.5 kD與47.2 kD的蛋白作為疫苗抗原候選蛋白,經(jīng)過分離、電洗脫純化得到這兩種布魯氏菌OMPs。采用α-D-甘露吡喃異硫氰酸苯酯對這兩種蛋白質抗原進行甘露糖修飾,以靶向體內抗原提呈功能最強的專職抗原提呈細胞—樹突狀細胞,再經(jīng)間苯二酚-硫酸法鑒定兩種蛋白質抗原糖基修飾效果。 采取方差分析比較布魯氏菌外膜蛋白糖基修飾后的免疫效果,以血清中抗體水平與淋巴細胞增殖能力作為檢測指標,隨機選取20只小鼠,并隨機分成A、B、C和D四組,分別肌注布魯氏菌S2商品疫苗、純化的布魯氏菌兩種混合外膜蛋白、甘露糖修飾化的布魯氏菌外膜蛋白和PBS。試驗結果表明:二免后4d、7d、10d ,A組、B組和C組間血清抗體水平差異不顯著(P0.05),但A組、B組、C組與D組相比差異極顯著(P0.01),具有統(tǒng)計學意義,表明布魯氏菌S2商品疫苗、布魯氏菌兩種混合外膜蛋白、修飾化的布魯氏菌外膜蛋白免疫小鼠后,血清抗體水平均顯著高于陰性對照組,明顯誘導了機體的體液免疫應答。由淋巴細胞增殖試驗表明:C組內的實驗組OD值/對照組OD值明顯高于A組、B組、D組。說明小鼠免疫甘露糖修飾化的布魯氏菌外膜蛋白在二免10d后,T淋巴細胞增殖能力明顯高于免疫布魯氏菌S2商品疫苗、純化的布魯氏菌兩種混合外膜蛋白與PBS對照組。表明甘露糖修飾化的布魯氏菌外膜蛋白在誘導機體的細胞免疫應答發(fā)揮了作用。以上實驗結果證實蛋白質經(jīng)糖基化修飾后有顯著免疫效果。 疫苗佐劑的選擇直接影響著疫苗的免疫效果,為了使疫苗的效果達到最佳,選擇了法國賽比克公司的四種佐劑Montanide ISA 71 VG、ESSAI ISA 206 CEL、Montanide IMS 251C VG和Montanide Gel 01 PR,利用糖基化布魯氏菌OMPs制作了四種不同佐劑的疫苗。由正交試驗篩選布魯氏菌外膜蛋白的配合佐劑,最佳免疫程序。結果顯示:抗原使用Montanide IMS 251C VG納米顆粒佐劑,免疫劑量為甘露糖基化修飾的布魯氏菌外膜蛋白X1μg,采取肌內注射,免疫三次這種組合,無論是在抗體產(chǎn)生趨勢還是血清IFN-γ產(chǎn)生水平為最佳組合。 完全隨機試驗、腹腔巨噬細胞吞噬試驗進一步檢驗靶向化布魯氏菌樹突狀細胞疫苗免疫效果,結果表明靶向化疫苗誘導了機體較強的免疫應答。安全性試驗結果表明:靶向化布魯氏菌外膜蛋白樹突狀細胞疫苗對6~8周齡BALB/c健康小鼠、妊娠小鼠均安全。 本研究的實驗結果為基于疫苗研究策略,有效預防布魯氏病奠定了基礎。
[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.

【學位授予單位】：河北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【引證文獻】

相關碩士學位論文 前1條

1 程佳;豬型布魯氏菌Omp31-BLS融合肽的誘導表達與多克隆抗體的制備[D];內蒙古科技大學;2012年

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本文編號：1409914