Deveropmental biotechnological analysis of host defense mechanisms against protozoan infection

宿主防御原虫感染机制的生物技术发展分析

• 批准号: 08306019
• 负责人: TOYODA Yutaka
• 金额: $ 12.03万
• 依托单位: OBIHIRO UNIVERSITY OF AGRICULTURE AND VETERINARY MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08306019/
• 关键词: Transgenic; macrophage; Scavenger receptor; Interferon; Babesia; Toxoplasma; Trypanosoma; mouse; インターフエロン; ネオスポーラ原虫

(l)Studies on gene knock-out miceExperiments on the host defense mechanisms against protozoan infection were performed using a macrophage scavenger receptor (SR) knock-out mice. As the SR is known to bind an extraordinary wide range of ligands including bacterial pathogens and mediate macrophage adhesion, it would be of interest to know the response of SR knock-out mice against protozoan infection. After intraperitoneal inoculation with Babesia microti, parasitemia was monitored daily by counting parasitized erythrocytes.It was found that the course of parasitemia was not different between SR-/- and SR+/+ mice but the decrease in packed cell volume at 16-30 days after infection was less obvious in SR-/- than in SR+/+ mice. Increase in spleen weight after B.microti infection was also smaller in SR-/- than in SR+/+ mice. Flow cytometric studies revealed that the percentages of Thy 1.2+ as well as CD4+ T cells in spleen remained relatively higher in SR-/- mice as compared to those in SR+/ … More + mice after infection. These results suggest that the SR knock-out mouse may become a useful model for the analysis of protozoan infection, especially with regard to the recovery processes, although it seems not to be directly involved in the defense mechanisms against B.microti infection.Studies using other types of knock-out mice, such as IFN gamma and iNOS, were also conducted.(2) Production of transgenic mice carrying protozoan geneSAG-i (p30), the major surface protein of Toxoplasma gondii, is considered as an important ligand in the process of host cell invasion. If this protein is produced by host cells and interfere with that of parasites, then the host will become resistant to parasite invasion, Or, it may become more susceptible to infection due to the lack of immune response against p30 antigen. To generate transgenic mice carrying p30 gene, a 3.3kb DNA fragments, containing p30 cDNA fused with CAG promoter, that has been shown to direct a ubiquitous expression of a reporter gene in transgenic mice, were microinjected into one of the pronuclei of one-cell embryos of C57BL/6J, BALB/c or F1 (C57BL/6J x C3H/He) mice. The embryos were transferred to the oviducts of pseudopregnant ICR mice, In the first series of experiment using inbred strains, two p30-positive founders (male C57BL/6J and female BALB/c) were obtained among 159 mice that developed from the injected eggs. Furthermore, we have obtained several P30-founder mice expressing the gene product and transmitting the gene to the next generation, by using F1 (C57BL/6J x C3H/He) embryos. As far as we are aware, this is the first transgenic mice bearing a protozoan gene derived from T.gondii. Less

（1）基因敲除小鼠的研究用巨噬细胞清道夫受体（SR）敲除小鼠进行宿主抗原生动物感染防御机制的实验。由于已知SR结合包括细菌病原体在内的非常广泛的配体并介导巨噬细胞粘附，因此了解SR敲除小鼠对原生动物感染的反应将是有意义的。结果表明，SR-/-和SR+/+小鼠的寄生虫血症过程无明显差异，但在感染后16-30天，SR-/-小鼠的红细胞压积下降较SR+/+小鼠明显。SR-/-小鼠感染B.microti后脾脏重量的增加也小于SR+/+小鼠。流式细胞术研究显示，与SR+/-小鼠相比，SR-/-小鼠脾脏中Thy 1.2+和CD 4 + T细胞的百分比保持相对较高。 ...更多信息 + 感染后的老鼠这些结果表明，SR基因敲除小鼠可能成为一个有用的模型，用于分析原生动物感染，特别是在恢复过程中，虽然它似乎没有直接参与防御机制对B.microti感染。研究使用其他类型的敲除小鼠，如IFN γ和iNOS，也进行了。(2)弓形虫表面蛋白SAG-1（p30）是弓形虫的主要表面蛋白，被认为是弓形虫入侵宿主细胞过程中的重要配体。如果宿主细胞产生这种蛋白质并干扰寄生虫的蛋白质，那么宿主将变得对寄生虫入侵具有抵抗力，或者由于缺乏针对p30抗原的免疫应答而变得更容易感染。为获得p30基因的转基因小鼠，将一个3.3kb的DNA片段（含p30 cDNA和CAG启动子）注射到C57 BL/6 J、BALB/c和F1（C57 BL/6 J × C3 H/He）小鼠的一个单细胞胚胎原核中。将胚胎移植到假孕ICR小鼠的输卵管中。在第一系列使用近交系的实验中，从159只注射卵发育的小鼠中获得了两个p30阳性的建立者（雄性C57 BL/6 J和雌性BALB/c）。此外，我们已经获得了几个P30-创始人小鼠表达的基因产物和传递基因的下一代，通过使用F1（C57 BL/6 J × C3 H/He）胚胎。据我们所知，这是第一个转基因小鼠携带原生动物基因来自T. gondii。少

项目成果

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