细粒棘球蚴谷胱甘肽s-转移酶免疫保护力观察及其免疫机制的研究

发布时间：2018-01-16 21:23

本文关键词：细粒棘球蚴谷胱甘肽s-转移酶免疫保护力观察及其免疫机制的研究　出处：《宁夏医科大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的在已克隆并成功构建细粒棘球蚴(Echinococcus granulosus,Eg)重组原核表达质粒pET-28a/GST的基础上,表达并纯化出重组蛋白Eg.GST,进行动物免疫保护力实验及免疫机制研究,以鉴定其疫苗的潜质。方法(1)重组表达质粒的诱导表达及纯化:IPTG诱导表达重组蛋白EgGST(rEgGST),经含Ni2+的His-bind树脂柱纯化rEgGST。(2)动物分组及免疫方案:选取132只雌性6周龄ICR小鼠随机分为3组即实验组、佐剂+PBS组和PBS组,每组44只;采用背部皮下多点注射,连续免疫3次,每次间隔两周;免疫小鼠10周后,每只小鼠用1500个活原头蚴腹腔注射进行攻击感染。分别于免疫前和攻击后的0周、2周、4周、6周、10周、18周、30周剖杀小鼠,并且眼眶取血分离血清。(3)rEgGST诱导小鼠的免疫保护力的计算:根据Dempster公式:免疫保护力(%)=(1-免疫组平均包囊数/对照组平均包囊数)×100㳠,计算免疫保护力(4)rEgGST诱导小鼠的免疫保护力及其机制研究:①通过ELISA方法对rEgGST诱导小鼠体液免疫指标的变化进行研究;②通过流式技术对rEgGST诱导小鼠细胞免疫指标的变化进行研究;③rEgGST诱导小鼠细胞因子变化的研究及MTT法检测小鼠脾淋巴细胞增殖情况。结果(1):将已经构建的基因工程菌株Eg.GST/pET-28a/BL21(DE3) plysS,表达并纯化出分子量约28kD的重组EgGST。(2):根据公式计算rEgGST能诱导小鼠产生89.39%的免疫保护力。(3)①rEgGST组小鼠在抗原免疫后和攻击感染后均产生高水平的IgG、低水平的IgG1和IgE,IgG2a、IgG2b和IgG3抗体水平与免疫前相比是升高的,提示IgG、IgG3、IgG2a和IgG2b可能参与保护性的免疫应答机制。②rEgGST组小鼠在攻击感染后不仅CD4+T细胞增加,CD8+T细胞也有轻度增加,CD4+/CD8+比值与PBS对照组比较有所降低;在攻击感染后30周rEgGST组的CD25+细胞和佐剂组与空白组相比差异具有显著性,而实验组与对照组之间无显著性差异。③rEgGST组小鼠在免疫后产生高水平的IL-2、IFN-γ和TNF-α,低水平的IL-4和IL-10;攻击感染后期IL-2、IFN-γ和TNF-α降低,IL-4和IL-10上升。提示该重组抗原以诱导Th1型免疫应答为主的反应;同时攻击感染后进行脾淋巴细胞增殖试验,MTT法检测证实rEgGST免疫的小鼠在rEgGST刺激下脾淋巴细胞增殖水平明显高于PBS对照组(P0.01)。结论( 1):成功表达并纯化出rEgGST。(2):rEgGST能诱导小鼠产生89.39%的免疫保护力,提示rEgGST蛋白能诱导机体产生较强的保护力,是一个极具潜力的抗包虫病疫苗候选分子。(3):rEgGST保护性免疫主要通过诱导宿主产生体液免疫应答和Th1型免疫应答,表明rEgGST是具有发展前途的抗包虫病候选疫苗。
[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- 纬 and TNF- 伪, and low levels of IL-4 and IL-10 after immunization. In the late stage of attack, IL-2n- 纬 and TNF- 伪 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.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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