重组全长副肌球蛋白在小鼠与水牛中杭日本血吸虫感染的保护力研究

发布时间：2019-06-22 15:59

【摘要】：日本血吸虫病是一种严重危害我国人民身体健康的寄生虫病。根据2006年的调查资料,我国现有血吸虫病人51万多,有6000多万人口受到血吸虫病的威胁。近年,我国血吸虫病防治以人畜同步化疗为主,消灭易感地带钉螺和加强健康教育为辅的综合防治措施。多年的防治实践证明,这一综合措施虽然有效,但效果难以巩固,不仅受到生态环境和防治力度变化的挑战,而且面临着人群化疗后再感染的困扰。单纯依赖化疗难以达到防治目标,鉴于疫苗在许多传染病控制中无可比拟的作用,研发血吸虫病疫苗,获取化疗短期效应与疫苗接种诱导的长期免疫预防作用相结合的双重效果,将是未来有效防治血吸虫病的主要奋斗目标。在我国,耕牛作为主要的保虫宿主和传染源在血吸虫病的流行过程中起着重要的传播作用。发展适合耕牛等主要保虫宿主的动物疫苗对血吸虫病的控制具有重要意义,此举不仅可通过控制传播降低人群血吸虫病流行率,而且为人用血吸虫病疫苗的研制提供一定的理论依据。血吸虫病疫苗的研究历时70余年,大体上经历了死疫苗、减毒活疫苗、基因工程疫苗、核酸疫苗研究阶段。作为血吸虫病疫苗候选分子之一的副肌球蛋白,在曼氏血吸虫或日本血吸虫的动物模型中均获得了不错的保护效果。相比其他血吸虫病疫苗候选分子,副肌球蛋白诱生的保护力较为突出,虫源性或重组副肌球蛋白分子片段,以及全长的DNA疫苗在小鼠、水牛等动物实验中的减虫率可以达到60%以上。然而,许多保护力实验中所用的副肌球蛋白为成虫的生化纯化产物,或大肠杆菌中的重组表达产物。生化纯化需要大量的虫源,而重组表达一直未能解决全长片段纯化困难、得率偏低的问题,长期以来,成为阻止其作为潜在疫苗快速推进到大规模研究,尤其是应用于大动物实验的障碍。免疫佐剂在调节免疫和提高疫苗效价方面具有重要作用,是疫苗获得理想效果的一条重要途径。良好的免疫佐剂不仅应该能够加强抗原的免疫原性和免疫保护效果,同时也应该不引起过敏反应或激发自身免疫反应等副作用。本研究使用的重组副肌球蛋白分子表达于大肠杆菌,采用改进的纯化方法后纯度能够达到95%以上,适于大规模制备与研究用途,重组蛋白结构和生物学特性与虫源性副肌球蛋白一致;先后使用多种佐剂与重组副肌球蛋白联合免疫小鼠,寻求最佳的疫苗组合;探索了ISA206佐剂与重组副肌球蛋白联合免疫水牛后对日本血吸虫感染的抵抗能力,以此来寻求适合于家畜(主要为耕牛)等保虫宿主的副肌球蛋白疫苗组合。本研究获得如下结果: 1.大规模重组表达的全长副肌球蛋白分子的纯度达到95%以上,其中LPS含量为0.07EU/ml,与虫源性副肌球蛋白分子具有相同的二级结构和生物学功能,后者包括与免疫球蛋白的结合特性以及与胶原物质的结合特性。 2.在昆明鼠与C57BL/6小鼠模型中,rSj97-Alhydrogel未能诱导理想的保护力。 3.在C57BL/6小鼠模型中,就减虫率与减卵率而言,与rSj97-FA组和rSj97-CpG-ODN组相比,rSj97-ISA206组对C57BL/6小鼠具有显著的保护效果,分别为45.3%(P0.01)与35.4%(P0.05),该结果提示,rSj97-ISA206具良好的抗日本血吸虫感染的保护效果。 4.在水牛模型中,rSj97-ISA206组相比ISA206佐剂组,所获减虫率为28.01%(P=0.443)、粪便虫卵减少率为28.03%(P=0.584);相比感染对照组,减虫率为16.08%(P=0.742)、肝脏减卵率为3.64%(P=0.963)、粪便减卵率为38.70%(P=0.462)。 5. rSj97在C57BL/6小鼠体内诱生出高水平特异性IgG2a与IgG1抗体,提示rSj97能诱导特异性Th1和Th2特征抗体亚类的产生,可能与诱导C57BL/6小鼠的保护力存在一定相关性。 6. rSj97免疫水牛后能诱生出高水平特异性IgG2与IgG1抗体,并诱生出Th1型优势应答,同时引起的体液免疫与细胞免疫应答可能是水牛产生保护力的因素之一。本研究进一步丰富了副肌球蛋白的保护效果研究,尤其是在大动物体内的研究,为构建适合于水牛的副肌球蛋白疫苗组合提供了重要的基础资料。
[Abstract]:Schistosomiasis japonica is a parasitic disease that is seriously harmful to the health of our people. According to the survey data of 2006, more than 550,000 of the present schistosomiasis has been made in China, and more than 60 million people have been threatened by the schistosomiasis. In recent years, the prevention and control of schistosomiasis in China is the main control measures for the prevention and control of the synchronous chemotherapy of the livestock and the livestock and the elimination of the Oncomelania hupensis and the strengthening of the health education. The years of prevention and control have proved that this comprehensive measure, though effective, is difficult to consolidate, not only is the challenge of the change of the ecological environment and the prevention and control, but also the problem of re-infection after the chemotherapy of the population. It is difficult to achieve the goal of prevention and cure by relying on chemotherapy alone, and in view of the incomparable role of the vaccine in the control of many infectious diseases, the double effects of the combination of the short-term effect of chemotherapy and the long-term immune prevention induced by the vaccine inoculation are obtained, It will be the main goal of the future effective control of the schistosomiasis. In our country, the cultivation of cattle as the main insect-protecting host and the source of infection plays an important role in the epidemic of schistosomiasis. The development of the animal vaccine suitable for the host of the main insect-protecting host such as the cultivated cattle is of great significance to the control of the schistosomiasis, which not only can reduce the prevalence rate of the schistosomiasis in the population by controlling the transmission, but also provides a certain theoretical basis for the development of the schistosomiasis vaccine for human use. The study of schistosomiasis vaccine has lasted for more than 70 years, and has generally undergone the study of dead vaccine, attenuated live vaccine, genetic engineering vaccine and nucleic acid vaccine. para. as the paramyosin of one of the candidate molecules of the schistosomiasis vaccine, a good protective effect is obtained in the animal models of the Schistosoma mansoni or the Schistosoma japonicum Compared with other schistosomiasis vaccine candidate molecules, the protection force of the paramyosin induced by the paramyosin is more prominent, the insect-derived or recombinant paramyosin molecule fragment, and the full-length DNA vaccine can reach 60% in animal experiments such as mice and buffalo. However, the secondary myosin used in many of the protection experiments is the biochemical purification product of the adult, or the recombinant expression in E. coli The biochemical purification requires a large number of entomogenous sources, and the recombinant expression has been unable to solve the problems of difficult purification and low yield of the full-length fragment, and has long been a barrier to the rapid advance of the full-length fragment to a large-scale study, in particular to a large animal experiment The immune adjuvant plays an important role in regulating immunity and improving the titer of the vaccine, and is a weight of the ideal effect of the vaccine. The good immune adjuvant not only can enhance the immunogenicity and the immune protective effect of the antigen, but also does not cause an allergic reaction or stimulate the self-immune reaction, and the like. The recombinant paramyosin molecule used in the study is expressed in E. coli, and the purity can reach more than 95% after the improved purification method, and is suitable for large-scale preparation and research purposes, the structure and biological characteristics of the recombinant protein and the worm-derived submuscle ball A combination of adjuvant and recombinant paramyosin was used to immunize mice, to find the best combination of the vaccine, and to explore the effect of the adjuvant of ISA206 and the recombinant paramyosin on the infection of Schistosoma japonicum. The ability to search for paramyosin phytophthora, which is suitable for the host of insect-protecting, such as cattle, and the like The combination of the seedlings. The present study was The results are as follows:1. The purity of the full-length paramyosin molecule expressed by the large-scale recombinant expression is over 95%, wherein the content of the LPS is 0.07 EU/ ml and the same secondary junction with the insect-derived paramyosin molecule Construction and biological functions, which include binding properties to immunoglobulins and with collagen the binding properties of the substance.2. In the Kunming mice and the C57BL/6 mouse model, rSj97-Hydrogel did not 3. Compared with rSj97-FA group and rSj97-CpG-ODN group, the rSj97-ISA206 group had significant protective effect on C57BL/6 mice compared with rSj97-FA group and rSj97-CpG-ODN group in the C57BL/6 mouse model. 4. Compared with the control group, the deinsectization rate was 16.08% (P = 0.742), the egg reduction rate of the liver was 3.64% (P = 0.963), and the egg reduction rate was 38. ..70% (P = 0.462). rSj97 induced a high level of specific IgG2a and IgG1 antibody in C57BL/6 mice, suggesting that rSj97 can induce the generation of specific Th1 and Th2-specific antibody subclasses, possibly related to the induction of C57BL/6 mice. 6. A high level of specific IgG2 and IgG1 antibodies can be induced by the rSj97 immune buffalo, and a Th1-type dominant response is induced, and humoral immunity and cellular immunity are induced. The response may be one of the factors that the buffalo produces a protective force. The study further enriched the study of the protective effect of paramyosin, especially in large animals, in order to build a suitable water buffalo
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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