Cloning of bovine hemoparasites and isolation of genetic markers for the parasite clones

牛血寄生虫的克隆和寄生虫克隆的遗传标记的分离

• 批准号: 09660325
• 负责人: ISHIHARA Chiaki
• 金额: $ 1.98万
• 依托单位: Rakuno-gakuen University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660325/
• 关键词: Babesia ovata; Theileria sergenti; Parasite cloning; Polymorphic genes; Genetic markers

Diseases caused by hemoparasitic protozoa, such as Babesia and Theileria, still remain to be a major threat to livestock industry worldwide. In order to facilitate the experimental studies on those hemoparasites, we have previously developed an animal model using SCID mice whose circulating red blood cells were substituted with bovine ones (Bo-RBC-SCID mice). In the present study, we used this animal model system to obtain parasite clones. RBC samples containing high parasitemia (more than 40%) were prepared by infection of Bo-RBC-SCID mice with B.ovata or T.sergenti. Single RBCs were individually picked under microscopy with a microcapillary tube, and each of them was inoculated into a Bo-RBC-SCID mouse. Parasite clones were established by repeating this procedure twice. We were able to isolate multiple parasite clones, whose characteristics appeared to be different from each other. To further define the individual parasite clones, we searched genetic markers which show polymorphism among parasite clones From the clone 11E of Babesia ovata, several genes were cloned and sequenced, including SpS-7, small subunit ribosomal RNA, actine, beta-tubulin, roptory associated protein-1, spherical body protein, merozoite surface protein, and elongation factor-1. Many of those genes were found to show polymorphism when they were used as the probe for southern blot analysis.In some genes, significant sequence variations were also seen between parasite clones. We were able to differentiate the individual parasite clones using combinations of several marker genes.The parasite clones and the polymorphic genetic markers obtained in the present study will serve as a useful experimental tool to study the diversified parasite population and possible genetic recombinational event occurring at the sexual stage in the vector ticks.

巴贝虫和泰勒虫等血液寄生原虫引起的疾病仍然是全世界畜牧业的主要威胁。为了便于对这些血液寄生虫的实验研究，我们之前使用SCID小鼠（其循环红细胞被牛细胞替代）开发了动物模型（Bo-RBC-SCID小鼠）。在本研究中，我们使用该动物模型系统来获得寄生虫克隆。通过用 B.ovata 或 T.sergenti 感染 Bo-RBC-SCID 小鼠来制备含有高寄生虫血症（超过 40%）的 RBC 样品。用微毛细管在显微镜下单独挑取单个红细胞，并将每个红细胞接种到Bo-RBC-SCID小鼠中。通过重复该过程两次来建立寄生虫克隆。我们能够分离出多个寄生虫克隆，它们的特征似乎彼此不同。为了进一步定义单个寄生虫克隆，我们搜索了显示寄生虫克隆之间多态性的遗传标记。从卵形巴贝虫克隆11E中，克隆并测序了几个基因，包括SpS-7、小亚基核糖体RNA、肌动蛋白、β-微管蛋白、ropory相关蛋白-1、球体蛋白、裂殖子表面蛋白和伸长因子-1。当这些基因被用作Southern印迹分析的探针时，发现许多基因表现出多态性。在一些基因中，在寄生虫克隆之间还发现了显着的序列变异。我们能够使用几个标记基因的组合来区分单个寄生虫克隆。本研究中获得的寄生虫克隆和多态性遗传标记将作为有用的实验工具来研究多样化的寄生虫种群和可能发生的基因重组事件。向量蜱的性阶段。

项目成果

Ishihara C.,Hiratai R.,et al.: "Mannan decelerate the clearance of human red blood cells in SCID mouse." Immunopharmacology. (in press).

Ishihara C.、Hiratai R. 等人：“甘露聚糖可减缓 SCID 小鼠中人红细胞的清除。”

Kim S-J., Tsuji M., Kubota S., Wei Q., Lee J-M,Ishihara C., and Onuma M.: "Sequence analysis of the major piroplasm surface protein gene of benign bovine Theileria parasites in East Asia." Int.J.Parasitol.28. 1219-1227 (1998)

Kim S-J.、Tsuji M.、Kubota S.、Wei Q.、Lee J-M、Ishihara C. 和 Onuma M.：“东亚良性牛泰勒虫寄生虫主要梨质表面蛋白基因的序列分析”。

Araia S., Tsuji M., Kim S-J., Nakadea T., Kanno Y., and Ishihara C.: "Babesia canis infection in canine-red blood cell-substituted SCID mice." Int.J.Parasitol.28. 1429-1435 (1998)

Araia S.、Tsuji M.、Kim S-J.、Nakadea T.、Kanno Y. 和 Ishihara C.：“犬红细胞替代 SCID 小鼠中的犬巴贝斯虫感染。”

Tsuji M., Arai S., Nakamura Y., Kim S.-J., Cho S.-H., He F.-Q., and Ishihara C.: "Preparation of antibodies directed to the Babesia ovata-or Theileria sergenti-parasitized erythrocytes." J.Vet.Med.Sci.60(12). 1321-1327 (1998)

Tsuji M.、Arai S.、Nakamura Y.、Kim S.-J.、Cho S.-H.、He F.-Q. 和 Ishihara C.：“针对卵形巴贝虫或泰勒虫的抗体的制备

Arai S., Tsuji M., Kim S.-J., Nakada K., Kirisawa R., and Ishihara C.: "Antigenic and genetic diversities of Babesia ovata in persistently infected cattle." J.Vet.Med.Sci.61(1). 73-76 (1999)

Arai S.、Tsuji M.、Kim S.-J.、Nakada K.、Kirisawa R. 和 Ishihara C.：“持续感染牛中卵形巴贝虫的抗原和遗传多样性。”