弓形虫ROP18-MIC2双基因疫苗对小鼠的免疫保护性研究

发布时间：2018-05-14 05:07

本文选题：弓形虫 + ROP18基因　；参考：《山东大学》2012年硕士论文

【摘要】：弓形虫是一种专性细胞内寄生机会致病原虫,分布广泛,可感染包括人类在内的大部分哺乳动物。人类对弓形虫普遍易感,孕妇感染后可通过胎盘垂直传染给胎儿,导致畸胎、死胎。弓形虫病也是最常见的机会性寄生虫病,该病成为免疫功能严重低下患者(如HIV/AIDS、恶性肿瘤患者)的主要死亡原因。迄今尚无治疗弓形虫病的特效药物,弓形虫疫苗和免疫保护性研究已逐渐引起了重视,并认为疫苗接种可能是预防弓形虫病的最佳策略。大量的动物和人体实验表明,DNA疫苗不仅可以预防,还可以治疗病原生物的感染。在弓形虫感染和包囊形成的防治过程中,疫苗是一种潜在高效经济的方法。由于弓形虫生活史较复杂,抗原成分具有发育阶段的特异性或差异性,因此在研究有关弓形虫疫苗的过程中,发展多种抗原组合、针对不同生活史发育阶段的复合多价疫苗是研究弓形虫疫苗过程中的一个发展方向与共识,这有助于克服单一抗原成分作为候选疫苗的不足。棒状体蛋白在虫体入侵和纳虫空泡的形成和修饰过程中起着重要作用,是重要的入侵和毒力作用因子。棒状体蛋白18(rhoptry protein18, ROP18)可以改变受感染的宿主细胞的信号传导,阻止宿主细胞凋亡,改变感染细胞的基因表达,介导受感染细胞出现病理损伤。在最新研究中,科学家们发现当弓形虫进入宿主细胞时,会生成一层保护膜包裹自身,使自己虽受细胞环境影响而不被杀死,ROP18的作用则是让宿主细胞某些蛋白失效而无法破坏这层保护膜。微线体蛋白2(microneme protein2, MIC2)属于血小管反应蛋白相关匿名蛋白家族,是虫体入侵宿主细胞过程中一个必需的入侵因子,并在弓形虫连接到宿主细胞的过程中发挥重要作用。然而,至今还未有研究对ROP18、MIC2作为抗弓形虫病疫苗的潜力进行评估和检测。本文利用一新型真核表达载体pBudCE4.1,此含有人巨细胞病毒(CMV)高效启动子、增强子序列,是一种外源基因在哺乳动物内高效表达的良好载体。该载体的特点是含有两侧分别有一些可插入的酶切位点,而且两侧分别都有各自的启动子。将ROP18与MIC2构建在此载体上,并作为DNA疫苗直接注射免疫小鼠来表达两种蛋白,以检测蛋白在小鼠体内诱导的免疫原性和安全性,并通过动物攻击实验,评价其免疫保护性。结果表明ROP18-MIC2双基因疫苗的效果显著优于ROP18或者MIC2单基因疫苗。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a parasitic opportunistic protozoa, which can infect most mammals, including humans. Human is susceptible to Toxoplasma gondii, pregnant women can be infected through the placenta vertical transmission to the fetus, resulting in teratogenesis, stillbirth. Toxoplasma gondii is also the most common opportunistic parasitic disease, which is the leading cause of death in patients with severe hypoimmunity (e.g. HIV / AIDS, malignant tumor). Up to now, there is no special drug to treat Toxoplasma gondii. Toxoplasma gondii vaccine and immune protection have been paid more and more attention, and it is considered that vaccination may be the best strategy to prevent Toxoplasma gondii from Toxoplasma gondii. A large number of animal and human experiments have shown that DNA vaccine can not only prevent, but also treat the infection of pathogenic organisms. Vaccine is a potentially efficient and economical method in the prevention and treatment of Toxoplasma gondii infection and cyst formation. Because the life history of Toxoplasma gondii is complicated and the antigen components have the specificity or difference of development stage, many kinds of antigen combinations have been developed in the research of Toxoplasma gondii vaccine. Multivalent vaccine for different stages of life cycle development is a development direction and consensus in the research of Toxoplasma gondii vaccine, which is helpful to overcome the shortage of single antigen as candidate vaccine. Rodlike proteins play an important role in the formation and modification of vacuoles and play an important role in invading and virulence. 18(rhoptry protein 18 (ROP18) can change the signal transduction of infected host cells, block the apoptosis of host cells, change the gene expression of infected cells, and mediate the pathological damage of infected cells. In a new study, scientists found that when Toxoplasma gondii enters the host cell, it forms a protective film that wraps itself. The effect of not killing ROP18, though affected by the cellular environment, is to invalidate certain proteins in the host cell and fail to destroy the protective membrane. Microline protein 2(microneme protein 2 (MIC2) belongs to the blood tubule reaction protein-associated anonymous protein family. It is an essential invading factor during the invasion of host cells and plays an important role in the process of connecting Toxoplasma gondii to host cells. However, no research has been done to evaluate and test the potential of ROP18 MIC2 as a vaccine against toxoplasmosis. In this paper, a new eukaryotic expression vector pBudCE4.1, which contains human cytomegalovirus (CMV) promoter and enhancer sequence, is a good vector for high expression of exogenous gene in mammals. The characteristic of the vector is that there are some insertable endonuclease sites on both sides of the vector, and each side has its own promoter. ROP18 and MIC2 were constructed on this vector and immunized mice with DNA vaccine directly to express the two proteins to detect the immunogenicity and safety of the proteins induced in mice and to evaluate their immune protection by animal attack experiments. The results showed that the effect of ROP18-MIC2 double gene vaccine was better than that of ROP18 or MIC2 single gene vaccine.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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