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

【摘要】：目的:對(duì)構(gòu)建的純化重組親肌肉抗原(rEg.myophilin)進(jìn)行免疫保護(hù)和免疫機(jī)制的研究,為包蟲(chóng)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(dá)、純化及疫苗制備。(2)動(dòng)物分組方案:選取111只小鼠隨即分為A、B對(duì)照組和實(shí)驗(yàn)組C組,分別對(duì)其進(jìn)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(duì)其對(duì)應(yīng)組進(jìn)行兩次加強(qiáng)免疫;最后用1500只細(xì)粒棘球蚴的原頭蚴腹腔攻擊感染。(3)免疫保護(hù)力計(jì)算:(免疫保護(hù)力計(jì)算公式:減囊率=1-實(shí)驗(yàn)組平均包囊數(shù)/對(duì)照組平均包囊數(shù)×100%)。(4)對(duì)小鼠進(jìn)行免疫保護(hù)機(jī)制的研究,①體液免疫抗體指標(biāo)的測(cè)定:從領(lǐng)取小鼠開(kāi)始按不同的時(shí)間點(diǎn),分別分批次對(duì)小鼠采血,進(jìn)行血清IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平的測(cè)定;②細(xì)胞免疫檢測(cè):從領(lǐng)取小鼠即開(kāi)始按不同的時(shí)間點(diǎn),分別分批次對(duì)小鼠取脾臟,并脾細(xì)胞刺激培養(yǎng),監(jiān)測(cè)其細(xì)胞因子IFN-γ、IL-4和IL-10的水平。結(jié)果:(1) rEg。myophilin能誘導(dǎo)小鼠產(chǎn)生86.11%以上的免疫保護(hù)力。(2)采集血清進(jìn)行ELISA血清抗體IgG、IgG1、IgG2a、IgG2b、IgG3和IgE以及細(xì)胞因子IFN-γ、IL-4和IL-10的檢測(cè),結(jié)果表明:抗體IgG、IgG1、IgG2a以及IgE,和細(xì)胞因子IFN-γ、IL-4分別在免疫之后出現(xiàn)了增高,對(duì)照組IL-10在攻擊感染后出現(xiàn)了明顯的增高,而在實(shí)驗(yàn)組則沒(méi)有相應(yīng)的增高。結(jié)論:(1)rEg.myophilin為極具應(yīng)用潛質(zhì)的疫苗候選分子。(2)對(duì)rEg.myophilin產(chǎn)生的免疫保護(hù)性可能機(jī)制:①抗體介導(dǎo)的體液免疫,IgG及其亞類IgG1和IgE介導(dǎo)的體液免疫,在抗細(xì)粒棘球蚴感染過(guò)程中發(fā)揮重要作用;②細(xì)胞免疫中,細(xì)胞因子水平變化顯示Th1類免疫應(yīng)答在試驗(yàn)組免疫保護(hù)中占優(yōu)勢(shì)作用,有利于機(jī)體抵御細(xì)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(jī)制得到了有效地阻遏,有利于機(jī)體的抗寄生蟲(chóng)。④體液免疫和細(xì)胞免疫相輔相成,相互促進(jìn),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(jī)制可能直接影響著IgE參與的嗜酸性粒細(xì)胞相關(guān)的抗寄生蟲(chóng)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(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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