弓形虫SAG1-MIC8复合基因疫苗对小鼠的免疫保护性研究

发布时间：2019-02-19 17:30

【摘要】： 刚地弓形虫(Toxoplasma gondii)是一种以猫科动物为终宿主的肠道球虫,呈世界性分布,其中间宿主范围较广,可感染包括人类在内的多种动物,引起人兽共患的弓形虫病。一般情况下,弓形虫感染终宿主或中间宿主感染后往往不产生明显的临床症状,但是在宿主免疫功能低下时,可致严重后果。慢性弓形虫病患者或者免疫抑制(如器官移植后长期应用免疫抑制剂)、免疫缺陷患者(如AIDS)合并感染弓形虫往往可见肺炎、心肌炎、脉络膜视网膜炎以及多系统脏器衰竭的发生;孕妇孕期感染弓形虫可导致流产、早产、畸胎、死胎等。由于在临床上弓形虫病的症状表现多样,给诊断治疗及预防等带来许多的不便,且临床上尚无十分理想的治疗药物,因此寻找有效的预防与治疗措施,研制安全有效的疫苗显得极为迫切。 DNA疫苗又称核酸疫苗,是继死疫苗、减毒或灭活疫苗、体外分离或基因工程制备的亚单位疫苗之后的又一类新型疫苗。大量的实验结果表明,DNA疫苗不仅具有预防疾病的作用,同时还具有治疗的作用。但是弓形虫生活史较复杂,抗原成分多,且具有发育阶段的特异性或差异性,因此成功的疫苗应针对多个特异性抗原,所产生的保护性免疫力需针对生活史中一个以上的感染阶段。动物和人体实验也表明,仅靠单种基因疫苗诱导的免疫效果并不理想,而复合基因疫苗诱导的各项免疫指标显著优于单价疫苗,有望成为未来基因疫苗中的研究方向。在本实验中,我们选用了SAG1和MIC8两种候选基因以构建复合DNA疫苗。 SAG1(main surface antigen 1)是弓形虫速殖子期特异的主要表面抗原之一,能刺激机体产生IgG、IgM、IgA等高效价的抗体以及细胞因子IFN-γ等杀死虫体,具有高度的免疫原性和免疫保护性,是弓形虫感染免疫诊断和疫苗开发的的主要候选抗原。除此之外,它还与粘附和穿入宿主细胞等功能有关。MIC8(microneme protein 8)是一个单基因拷贝序列,无内含子,它所编码的蛋白是MIC3蛋白的护送蛋白,在速殖子期和缓殖子期都有表达,可帮助MIC3蛋白到达微线体并分泌出胞。最新研究显示,当MIC8蛋白不存在的时候,虫体不能与宿主细胞形成接触融合区域,使得侵入宿主细胞过程受阻,这种由MIC8参与形成的细胞融合区域非常重要,不可被其他的微线体蛋白来替代,从而表明MIC8是一种新颖的,功能独特的蛋白。另外,MIC8蛋白参与的信号级联放大反应,可致棒状体蛋白的分泌。本研究成功的将SAG1和MIC8两个基因构建在同一个表达载体pcDNA3.1上,通过肌注的方式免疫C57BL/6J小鼠,观察复合基因疫苗的免疫效果。结果表明SAG1-MIC8复合基因疫苗的效果显著优于SAG1或者MIC8单基因疫苗。此外,在小鼠体内没有检测到高浓度的细胞因子白介素4(IL-4),且各组间浓度差别无统计学意义,因而不能确定是否有Th2型免疫反应参与了免疫应答。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) (Toxoplasma gondii) is a kind of intestinal coccidiosis with cat family as its final host. It has a wide range of intermediate hosts and can infect a variety of animals, including human beings, and cause zoonotic toxoplasmosis (Toxoplasma gondii). In general, Toxoplasma gondii infection with terminal host or intermediate host infection often does not produce obvious clinical symptoms, but when host immune function is low, it can lead to serious consequences. Patients with chronic toxoplasmosis or immunosuppression (such as long-term use of immunosuppressive agents after organ transplantation), immunodeficient patients (such as AIDS) with Toxoplasma gondii often have pneumonia, myocarditis, Choroidal retinitis and multiple system organ failure; Pregnant women infected with Toxoplasma gondii can lead to abortion, premature delivery, teratogenesis, stillbirth, etc. Because the symptoms of Toxoplasma gondii are various in clinic, it brings many inconveniences to diagnosis, treatment and prevention, and there is no ideal therapeutic drug in clinic, so we seek effective prevention and treatment measures. It is urgent to develop a safe and effective vaccine. DNA vaccine, also called nucleic acid vaccine, is a new type of vaccine after dead vaccine, attenuated or inactivated vaccine, subunit vaccine prepared by isolation or genetic engineering in vitro. A large number of experimental results show that DNA vaccine not only can prevent disease, but also has therapeutic effect. However, the life history of Toxoplasma gondii is more complicated, with many antigen components, and has the specificity or difference of development stage, so the successful vaccine should be aimed at many specific antigens. The protective immunity generated should be targeted at more than one stage of infection in the life cycle. Animal and human experiments also show that the immune effect induced by single gene vaccine is not satisfactory, and the immune indexes induced by compound gene vaccine are significantly better than that of monovalent vaccine, which is expected to become the research direction of gene vaccine in the future. In this study, we selected two candidate genes, SAG1 and MIC8, to construct compound DNA vaccine. SAG1 (main surface antigen 1) is one of the major surface antigens specific to Toxoplasma gondii tachyzoites. It can stimulate the production of high titer antibodies, such as IgG,IgM,IgA, and the cytokine IFN- 纬 to kill the parasites, so it has high immunogenicity and immunity protection. Toxoplasma gondii infection is the main candidate antigen for immune diagnosis and vaccine development. In addition, it is related to the function of adhesion and penetration into host cells. MIC8 (microneme protein 8) is a single gene copy sequence without introns. It encodes an escort protein of MIC3 protein, which is expressed in both tachyzoites and bradyzoites. It can help the MIC3 protein to reach the microline and secrete the cells. New research shows that when MIC8 protein does not exist, the parasite can't form contact fusion area with host cells, which prevents the invading of host cells. This kind of cell fusion region which is formed by MIC8 is very important. MIC8 is a novel and functional protein that cannot be replaced by other microsomal proteins. In addition, MIC8 protein involved in signal cascade amplification reaction, can induce the secretion of rodlike proteins. In this study, SAG1 and MIC8 genes were successfully constructed on the same expression vector pcDNA3.1, and the immunized C57BL/6J mice were immunized by intramuscular injection to observe the immune effect of the composite gene vaccine. The results showed that the effect of SAG1-MIC8 compound gene vaccine was better than that of SAG1 or MIC8 single gene vaccine. In addition, no high concentration of cytokine interleukin-4 (IL-4) was detected in mice, and there was no significant difference in the concentrations among the groups. Therefore, it was not possible to determine whether the Th2 type immune response was involved in the immune response.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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