本文關(guān)鍵詞：細(xì)粒棘球蚴谷胱甘肽s-轉(zhuǎn)移酶免疫保護(hù)力觀察及其免疫機(jī)制的研究　出處：《寧夏醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 細(xì)粒棘球蚴 谷胱甘肽s-轉(zhuǎn)移酶 免疫保護(hù)力 免疫機(jī)制

【摘要】：目的在已克隆并成功構(gòu)建細(xì)粒棘球蚴(Echinococcus granulosus,Eg)重組原核表達(dá)質(zhì)粒pET-28a/GST的基礎(chǔ)上,表達(dá)并純化出重組蛋白Eg.GST,進(jìn)行動(dòng)物免疫保護(hù)力實(shí)驗(yàn)及免疫機(jī)制研究,以鑒定其疫苗的潛質(zhì)。方法(1)重組表達(dá)質(zhì)粒的誘導(dǎo)表達(dá)及純化:IPTG誘導(dǎo)表達(dá)重組蛋白EgGST(rEgGST),經(jīng)含Ni2+的His-bind樹脂柱純化rEgGST。(2)動(dòng)物分組及免疫方案:選取132只雌性6周齡ICR小鼠隨機(jī)分為3組即實(shí)驗(yàn)組、佐劑+PBS組和PBS組,每組44只;采用背部皮下多點(diǎn)注射,連續(xù)免疫3次,每次間隔兩周;免疫小鼠10周后,每只小鼠用1500個(gè)活原頭蚴腹腔注射進(jìn)行攻擊感染。分別于免疫前和攻擊后的0周、2周、4周、6周、10周、18周、30周剖殺小鼠,并且眼眶取血分離血清。(3)rEgGST誘導(dǎo)小鼠的免疫保護(hù)力的計(jì)算:根據(jù)Dempster公式:免疫保護(hù)力(%)=(1-免疫組平均包囊數(shù)/對(duì)照組平均包囊數(shù))×100㳠,計(jì)算免疫保護(hù)力(4)rEgGST誘導(dǎo)小鼠的免疫保護(hù)力及其機(jī)制研究:①通過(guò)ELISA方法對(duì)rEgGST誘導(dǎo)小鼠體液免疫指標(biāo)的變化進(jìn)行研究;②通過(guò)流式技術(shù)對(duì)rEgGST誘導(dǎo)小鼠細(xì)胞免疫指標(biāo)的變化進(jìn)行研究;③rEgGST誘導(dǎo)小鼠細(xì)胞因子變化的研究及MTT法檢測(cè)小鼠脾淋巴細(xì)胞增殖情況。結(jié)果(1):將已經(jīng)構(gòu)建的基因工程菌株Eg.GST/pET-28a/BL21(DE3) plysS,表達(dá)并純化出分子量約28kD的重組EgGST。(2):根據(jù)公式計(jì)算rEgGST能誘導(dǎo)小鼠產(chǎn)生89.39%的免疫保護(hù)力。(3)①rEgGST組小鼠在抗原免疫后和攻擊感染后均產(chǎn)生高水平的IgG、低水平的IgG1和IgE,IgG2a、IgG2b和IgG3抗體水平與免疫前相比是升高的,提示IgG、IgG3、IgG2a和IgG2b可能參與保護(hù)性的免疫應(yīng)答機(jī)制。②rEgGST組小鼠在攻擊感染后不僅CD4+T細(xì)胞增加,CD8+T細(xì)胞也有輕度增加,CD4+/CD8+比值與PBS對(duì)照組比較有所降低;在攻擊感染后30周rEgGST組的CD25+細(xì)胞和佐劑組與空白組相比差異具有顯著性,而實(shí)驗(yàn)組與對(duì)照組之間無(wú)顯著性差異。③rEgGST組小鼠在免疫后產(chǎn)生高水平的IL-2、IFN-γ和TNF-α,低水平的IL-4和IL-10;攻擊感染后期IL-2、IFN-γ和TNF-α降低,IL-4和IL-10上升。提示該重組抗原以誘導(dǎo)Th1型免疫應(yīng)答為主的反應(yīng);同時(shí)攻擊感染后進(jìn)行脾淋巴細(xì)胞增殖試驗(yàn),MTT法檢測(cè)證實(shí)rEgGST免疫的小鼠在rEgGST刺激下脾淋巴細(xì)胞增殖水平明顯高于PBS對(duì)照組(P0.01)。結(jié)論( 1):成功表達(dá)并純化出rEgGST。(2):rEgGST能誘導(dǎo)小鼠產(chǎn)生89.39%的免疫保護(hù)力,提示rEgGST蛋白能誘導(dǎo)機(jī)體產(chǎn)生較強(qiáng)的保護(hù)力,是一個(gè)極具潛力的抗包蟲病疫苗候選分子。(3):rEgGST保護(hù)性免疫主要通過(guò)誘導(dǎo)宿主產(chǎn)生體液免疫應(yīng)答和Th1型免疫應(yīng)答,表明rEgGST是具有發(fā)展前途的抗包蟲病候選疫苗。
[Abstract]:Objective to construct the recombinant prokaryotic expression plasmid pET-28a/GST of Echinococcus granulosus Eg. The recombinant protein Eg.GST was expressed and purified. Methods the recombinant expression plasmid was induced to express and purify the recombinant protein EgGSTrEgGST induced by 1% IPTG. Purification of rEgGST. 2 by His-bind resin column containing Ni2): 132 female 6-week-old ICR mice were randomly divided into 3 groups: experimental group. Adjuvant PBS group and PBS group, 44 rats in each group; The patients were injected subcutaneously with multiple points in the back and immunized continuously for 3 times, with a two-week interval. After 10 weeks of immunization, each mouse was injected with 1500 live protocercariae into the abdominal cavity to infect the mice. The mice were infected by aggressive infection at 0 weeks before immunization and 0 weeks after the attack, respectively, at 2 weeks, 4 weeks, 10 weeks and 18 weeks, respectively, before and after immunization. Mice were dissected for 30 weeks. And the calculation of immune protection ability of mice induced by rEgGST-induced by serum from orbital blood: according to Dempster formula: immuno-protective effect of GST-induced mice (. 1- the average number of cysts in immunized group and the average number of cysts in control group) 脳 100? , to calculate the immune protection ability of mice induced by rEgGST and its mechanism; to study the changes of humoral immune indexes induced by rEgGST in mice by ELISA method. (2) the changes of cellular immune indexes induced by rEgGST in mice were studied by flow cytometry. Study on the changes of cytokines induced by 3rEgGST and MTT assay to detect the proliferation of splenic lymphocytes in mice. : the genetically engineered strain Eg.GST-pET-28a / BL21DE3 plysS was constructed. Expression and purification of Recombinant EgGST.GST2 with a molecular weight of about 28kD: according to the formula, we calculate that rEgGST can induce mice to produce an immuno-protective power of 89.39%. 1rEgGST group produced high level of IgG after antigen immunization and attack infection. The low levels of IgG2b and IgG2b and IgG3 antibody levels were higher than those before immunization, suggesting IgG3. IgG2a and IgG2b may be involved in the protective immune response mechanism. 2rEgGST group not only increased CD4 T cells but also slightly increased CD8 T cells after attack. The ratio of CD4 / CD8 was lower than that of PBS control group. There were significant differences in CD25 cells and adjuvants between the rEgGST group and the blank group 30 weeks after the attack. However, there was no significant difference between the experimental group and the control group. 3rEgGST group produced high levels of IL-2 IFN- 緯 and TNF- 偽, and low levels of IL-4 and IL-10 after immunization. In the late stage of attack, IL-2n- 緯 and TNF- 偽 decreased the increase of IL-4 and IL-10, suggesting that the recombinant antigen could induce Th1 type immune response. Spleen lymphocyte proliferation test was carried out after attacking infection at the same time. MTT assay confirmed that the proliferation level of splenic lymphocytes in mice immunized with rEgGST was significantly higher than that in PBS control group (P 0.01). Conclusion (1). REgGST.(2):rEgGST was successfully expressed and purified to induce immune protection of 89.39% in mice. These results suggest that rEgGST protein can induce strong protective effect. It is a potential candidate for vaccine against echinococcosis. It is mainly by inducing humoral immune response and Th1 type immune response of host. REgGST is a promising candidate vaccine against hydatid disease.
【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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