血吸虫果糖二磷酸醛缩酶的表达、纯化及诱导Th2免疫偏移的研究
发布时间：2018-08-14 13:18
【摘要】：背景：血吸虫病是由联合国开发计划署、世界银行和世界卫生组织热带病特别规划署共同倡议防治的一种主要的热带疾病。在非洲、南美洲以及亚洲等地区流行,血吸虫病是一项严重的公共卫生问题。全球约有2亿人感染血吸虫,1.2亿人有典型症状。血吸虫感染早期,宿主呈现Th1优势应答,随后出现Th2免疫偏移,其负向免疫调控的机制至今尚未明了。本课题通过表达、纯化虫源性糖蛋白果糖二磷酸醛缩酶(SjFBPA),感染小鼠和刺激细胞,在体外研究SjFBPA诱导Th2免疫应答的机制,对揭示虫体免疫逃避、宿主免疫耐受、免疫偏移等的分子机制的研究有重要意义。目的：分析日本血吸虫感染以及虫源性糖蛋白果糖二磷酸醛缩酶(SjFBPA)刺激后小鼠脾脏细胞中IL-4、CD8α因子的表达量的动态变化,并探讨SjFBPA与Th2免疫应答的关系。方法：本次实验首先通过转化将连接产物转入感受态细胞,然后小量表达菌株,电泳检测单克隆成功表达。再通过大量摇菌表达蛋白后进行纯化,得到大量高纯度的FBPA蛋白。构建血吸虫感染小鼠模型,于模型建立后设立FBPA蛋白刺激对照组。通过FCM检测IL-4、CD8α因子表达水平以及动物模型病理标本的改变。对FBPA蛋白诱导Th2免疫偏移的机制进行全面分析。结果：(1)通过转化后成功培养出单克隆表达菌落。(2)大量表达、纯化后可得到浓度达0.5mg/ml的FBPA蛋白。(3)血吸虫感染8W后小鼠脾脏细胞悬液的IL-4、CD8α因子表达量与正常对照组小鼠脾脏细胞悬液的IL-4、CD8α因子表达量比较发生明显变化,FBPA蛋白刺激后的小鼠脾脏细胞悬液的IL-4、CD8α因子表达量与正常对照组小鼠脾脏细胞悬液的IL-4、CD8α因子表达量比较也发生了明显变化,差异具有统计学意义(P0.05)。结论：本实验研究结果显示虫源性果糖二磷酸醛缩酶(SjFBPA)属于包涵体表达蛋白,通过转化方式可以得到单克隆表达菌,纯化可得到高浓度(0.5mg/ml)蛋白。SjFBPA喇激小鼠脾脏细胞悬液后细胞因子的变化与血吸虫感染后的小鼠脾脏细胞悬液变化趋势相同。DC细胞分泌因子CD8α的变化趋势也相同。可得出SjFBPA在血吸虫感染中具有诱导Th2免疫偏移的作用,DC细胞在此过程中参与了重要的免疫反应。
[Abstract]:Background: schistosomiasis is a major tropical disease under the joint initiative of the United Nations Development Programme (UNDP), the World Bank and the World Health Organization (WHO) Special Programme on Tropical Diseases. Schistosomiasis is a serious public health problem in Africa, South America and Asia. About 200 million people worldwide are infected with schistosomiasis and 120 million have typical symptoms. In the early stage of Schistosoma japonicum infection, the host presented a dominant response to Th1, followed by a Th2 immune shift. The mechanism of negative immune regulation has not been clarified. In this study, the expression and purification of arbuscular glycoprotein fructose diphosphate aldolase (SjFBPA),) were used to infect mice and stimulate cells. The mechanism of Th2 immune response induced by SjFBPA was studied in vitro. It is important to study the molecular mechanism of immune shift. Aim: to analyze the dynamic changes of IL-4 / CD8 伪 expression in spleen cells of mice after infection of Schistosoma japonicum and (SjFBPA) stimulation of insect-derived glycoprotein fructose diphosphate aldolase (SjFBPA), and to explore the relationship between SjFBPA and Th2 immune response. Methods: in this experiment, the ligation product was first transformed into the receptive cells, then expressed in a small number of strains, and the monoclonal expression was detected by electrophoresis. A large number of high purity FBPA proteins were obtained after purification by a large number of expressed proteins. The model of Schistosoma japonicum infected mice was established, and the control group was stimulated by FBPA protein after the establishment of the model. The expression level of IL-4 and CD8 伪 and the pathological changes of animal model were detected by FCM. The mechanism of Th2 immune migration induced by FBPA protein was analyzed. Results: (1) Monoclonal expression colonies were successfully cultured after transformation. After purification, FBPA protein with concentration up to 0.5mg/ml was obtained. (3) the expression of IL-4 and CD8 伪 in spleen cell suspension of mice infected with Schistosoma japonicum was significantly different from that in normal control mice. (3) the expression of IL-4 and CD8 伪 in spleen cell suspension of mice infected with Schistosoma japonicum was significantly changed after infection with Schistosoma japonicum for 8 weeks. The expression of IL-4 and CD8 伪 in splenic cell suspension of stimulated mice was significantly different from that in normal control group. The difference was statistically significant (P0.05). Conclusion: the results of this study showed that the insect-derived fructose diphosphate aldolase (SjFBPA) belonged to the inclusion body expression protein, and monoclonal expression bacteria could be obtained by transformation. The changes of cytokines in spleen cell suspension of SjFBPA-stimulated mice were similar to those of mice infected with Schistosoma japonicum. The change trend of CD8 伪 secreted by DC cells was the same as that in mice infected with Schistosoma japonicum. It can be concluded that SjFBPA can induce Th2 immune deviation in schistosomiasis infection. DC cells participate in the important immune response in the process of Schistosoma japonicum infection.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R532.21
本文编号：2182977
[Abstract]:Background: schistosomiasis is a major tropical disease under the joint initiative of the United Nations Development Programme (UNDP), the World Bank and the World Health Organization (WHO) Special Programme on Tropical Diseases. Schistosomiasis is a serious public health problem in Africa, South America and Asia. About 200 million people worldwide are infected with schistosomiasis and 120 million have typical symptoms. In the early stage of Schistosoma japonicum infection, the host presented a dominant response to Th1, followed by a Th2 immune shift. The mechanism of negative immune regulation has not been clarified. In this study, the expression and purification of arbuscular glycoprotein fructose diphosphate aldolase (SjFBPA),) were used to infect mice and stimulate cells. The mechanism of Th2 immune response induced by SjFBPA was studied in vitro. It is important to study the molecular mechanism of immune shift. Aim: to analyze the dynamic changes of IL-4 / CD8 伪 expression in spleen cells of mice after infection of Schistosoma japonicum and (SjFBPA) stimulation of insect-derived glycoprotein fructose diphosphate aldolase (SjFBPA), and to explore the relationship between SjFBPA and Th2 immune response. Methods: in this experiment, the ligation product was first transformed into the receptive cells, then expressed in a small number of strains, and the monoclonal expression was detected by electrophoresis. A large number of high purity FBPA proteins were obtained after purification by a large number of expressed proteins. The model of Schistosoma japonicum infected mice was established, and the control group was stimulated by FBPA protein after the establishment of the model. The expression level of IL-4 and CD8 伪 and the pathological changes of animal model were detected by FCM. The mechanism of Th2 immune migration induced by FBPA protein was analyzed. Results: (1) Monoclonal expression colonies were successfully cultured after transformation. After purification, FBPA protein with concentration up to 0.5mg/ml was obtained. (3) the expression of IL-4 and CD8 伪 in spleen cell suspension of mice infected with Schistosoma japonicum was significantly different from that in normal control mice. (3) the expression of IL-4 and CD8 伪 in spleen cell suspension of mice infected with Schistosoma japonicum was significantly changed after infection with Schistosoma japonicum for 8 weeks. The expression of IL-4 and CD8 伪 in splenic cell suspension of stimulated mice was significantly different from that in normal control group. The difference was statistically significant (P0.05). Conclusion: the results of this study showed that the insect-derived fructose diphosphate aldolase (SjFBPA) belonged to the inclusion body expression protein, and monoclonal expression bacteria could be obtained by transformation. The changes of cytokines in spleen cell suspension of SjFBPA-stimulated mice were similar to those of mice infected with Schistosoma japonicum. The change trend of CD8 伪 secreted by DC cells was the same as that in mice infected with Schistosoma japonicum. It can be concluded that SjFBPA can induce Th2 immune deviation in schistosomiasis infection. DC cells participate in the important immune response in the process of Schistosoma japonicum infection.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R532.21
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