本文選題：細粒棘球蚴 + 重組親肌肉抗原��； 參考：《寧夏醫(yī)科大學》2010年碩士論文

【摘要】：目的:對構(gòu)建的純化重組親肌肉抗原(rEg.myophilin)進行免疫保護和免疫機制的研究,為包蟲病分子疫苗的研制提供新的候選分子。方法:(1)疫苗制備:重組Eg.myophlilin/pET-28a工程菌株的大量表達、純化及疫苗制備。(2)動物分組方案:選取111只小鼠隨即分為A、B對照組和實驗組C組,分別對其進行皮下PBS、PBS+佐劑和PBS加佐劑加候選疫苗的注射,之后分別用同樣的試劑對其對應組進行兩次加強免疫;最后用1500只細粒棘球蚴的原頭蚴腹腔攻擊感染。(3)免疫保護力計算:(免疫保護力計算公式:減囊率=1-實驗組平均包囊數(shù)/對照組平均包囊數(shù)×100%)。(4)對小鼠進行免疫保護機制的研究,①體液免疫抗體指標的測定:從領(lǐng)取小鼠開始按不同的時間點,分別分批次對小鼠采血,進行血清IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平的測定;②細胞免疫檢測:從領(lǐng)取小鼠即開始按不同的時間點,分別分批次對小鼠取脾臟,并脾細胞刺激培養(yǎng),監(jiān)測其細胞因子IFN-γ、IL-4和IL-10的水平。結(jié)果:(1) rEg。myophilin能誘導小鼠產(chǎn)生86.11%以上的免疫保護力。(2)采集血清進行ELISA血清抗體IgG、IgG1、IgG2a、IgG2b、IgG3和IgE以及細胞因子IFN-γ、IL-4和IL-10的檢測,結(jié)果表明:抗體IgG、IgG1、IgG2a以及IgE,和細胞因子IFN-γ、IL-4分別在免疫之后出現(xiàn)了增高,對照組IL-10在攻擊感染后出現(xiàn)了明顯的增高,而在實驗組則沒有相應的增高。結(jié)論:(1)rEg.myophilin為極具應用潛質(zhì)的疫苗候選分子。(2)對rEg.myophilin產(chǎn)生的免疫保護性可能機制:①抗體介導的體液免疫,IgG及其亞類IgG1和IgE介導的體液免疫,在抗細粒棘球蚴感染過程中發(fā)揮重要作用;②細胞免疫中,細胞因子水平變化顯示Th1類免疫應答在試驗組免疫保護中占優(yōu)勢作用,有利于機體抵御細粒棘球蚴的感染;③該疫苗候選分子的注入使得免疫抑制機制得到了有效地阻遏,有利于機體的抗寄生蟲。④體液免疫和細胞免疫相輔相成,相互促進,表現(xiàn)在:IL-4在IgE亞型類別轉(zhuǎn)換中起到了關(guān)鍵的作用,這一機制可能直接影響著IgE參與的嗜酸性粒細胞相關(guān)的抗寄生蟲感染。
[Abstract]:Objective: to study the immune protection and immunological mechanism of the purified recombinant muscular antigen rEg.myophilin in order to provide a new candidate for the development of hydatidosis vaccine. Methods 1) Vaccine preparation: recombinant Eg.myophlilin/pET-28a engineering strain was expressed, purified and vaccine preparation. 2) Animal grouping: 111 mice were randomly divided into two groups: control group and experimental group C, the control group was divided into two groups, the control group and the control group, and the control group was divided into two groups, the control group and the control group. They were injected with subcutaneous PBS- PBS adjuvant and PBS plus adjuvant plus candidate vaccine respectively, and then their corresponding groups were immunized twice with the same reagent. At last, the immune protective mechanism of 1500 echinococcus granulosus was studied by calculating the immune protection ability of the mice by using the immune protection power (the formula of the immune protective power: the ratio of reducing the cysts was 1- the average number of the cysts in the experimental group and the average number of the cysts in the control group 脳 100th), and the immune protection mechanism of the mice was studied by using the immune protection ability of 1500 echinococcus granulosus. 1 determination of humoral immune antibody index: blood samples were collected from mice at different time points from receiving mice, and the levels of IgG2G1 and IgE in serum were determined by different time points, and the levels of IgG2G2bG3 and IgE were detected by different time points from the receiving mice, and the blood samples were collected from each group of mice at different time points, and the levels of IgG2bG3 and IgG2bG3 in the sera of the recipient mice were determined at different time points. Spleen was collected from mice in different batches, and spleen cells were stimulated and cultured to monitor the levels of IL-4 and IL-10. Results rEg.myophilin could induce mice to produce more than 86.11% of immuno-protective power.) Serum samples were collected to detect IgG3 and IgE, and cytokine IFN- 緯 IL-4 and IL-10. The results showed that the levels of IgG2a, IgE, and cytokine IFN- 緯 IL-4 were increased after immunization. The IL-10 of the control group was significantly increased after infection, but not in the experimental group. Conclusion the possible mechanism of immune protection of rEg.myophilin induced by 1: 1 antibody against rEg.myophilin and its subclasses of IgG1 and IgE mediated by IgG1 and IgE may be the possible mechanism of immuno-protective mechanism of IgG1. Myophilin is a potential vaccine candidate molecule. In the process of anti-infection of Echinococcus granulosus, the change of cytokine level showed that the immune response of Th1 was dominant in the immune protection of the experimental group, which was beneficial to resist the infection of echinococcus granulosus. (3) the injection of candidate vaccine molecules makes the immunosuppressive mechanism effectively repress, which is beneficial to the body's anti-parasite .4 humoral immunity and cellular immunity, which complement each other and promote each other. This mechanism may directly affect the anti-parasitic infection associated with eosinophilic granulocytes involved in IgE.
【學位授予單位】：寧夏醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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