The mechanism of site specificity of parasite : Study with avian coccidiosis

寄生虫位点特异性的机制：禽球虫病研究

• 批准号: 07456145
• 负责人: BABA Eiichiroh
• 金额: $ 4.61万
• 依托单位: Osaka Prefecture University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456145/
• 关键词: Eimeira; Chicken monoclonal antibody; Carbohydrates; Apical complex; Westem Blotting analysis; 間接蛍光抗体法; ウェスタンブロッテイング; 鶏モノクローナル抗体; 感染防御; D-galactose; コ-ノイド; 鶏モクローナル抗体; 共通抗原性; 免疫組織学的手法; 分子生物学的手法

Invasive protozoa have been known to recognize specific sugar residues of host cell surface and use the apical complex at the penetration into cells. At the first step of this series of study, the effects of carbohydrates on the penetration of Eimeria tenella sporozoites into cultured cells were investigated. The penetration of sporozoites was sppressed when pretreated with peanut lectin that specifically recognizes D-galactos residues at a concentration of 50 ml.ml. At the second step, to producing chicken monoclonal antibody against apical complex of Eimeria to get more knowlede during the invasion. Many stable chicken hybridoma secreting a monoclonal antibody (mAb) that detect the apical complex of Eimeria acervulina sporozoites has been developed by fusing a thymidine kinase (TK) -deficient chicken myeloma with spleen cells from chickens immunized with sporozoite antigen. One of the mAbs, designated as 6D-12-G10, recognized the apical complex of a sporozoite of 20-21 kDa molecular … More mass on western blots. Immunoelectron microscopic examination revealed that mAb 6D-12-G10 stained the conoid antigen. Furthermore, mAb 6D-12-G10 inhibited the invasion of sporozoites into CD8+ T cells in vitro. At the next step, the species-specificity and cross-reactivity of chicken developed against E.acervulina (EA) sporozoite were determined by confocal laser scanning microscopy and Western blotting analysis using the sporozoites of seven different avian Eimeria species such as EA,E.brunetti (EB), E.maxima (EM), E.mitis, E.necatrix, E.praecox and E.tenella. The five different mAbs, named 8E-1, HE-4,8D-2,5D-11 and 8C-3 were used in this study. In the immunofluorescent examination, these mAbs showed similar reactivity on the apical complex of the sporozoite. One of these mAbs, 8E-1 cross-reacted with all Eimeria species that were examined, HE-4 and 8D-2 mAbs reacted with EA and EB,5D-11 mAb reacted with EA and EM,8C-3 mAb reacted with only EA.In Western blot analysis using sonicated aporozoites of the seven of the Eimeria species as antigen, all mAbs recognized multiple bands ; the predominant bands of mAbs had molecular weights of approximately 32,43 and 260 kilodaltons. The present results of specific sugar residues of host cell surface suggested that D-galactose residues on E.tenella sporozoite surfaces and the lectin-like receptors that recognize D-galactose of host cells are important factors for penetration.Those monolconal antibodies might help more clear cut results of parasites invasion into the host cell. And also we might continue to study the relationship between the antigens which were recognized by mAbs and sugar residues. The availability of a technique to develop chicken mAbs should greatly enhance our ability to study the role of individual molecules involved in attachment, invasion, and motility. These molecules may prove to be important and novel targets for immunological and pharmacological therapy against coccidial infection. Less

已知入侵原生动物识别宿主细胞表面的特定糖残基，并在穿透细胞时使用顶端复合体。在这一系列研究的第一步，研究了碳水化合物对柔嫩艾美耳球虫子孢子穿透培养细胞的影响。当花生凝集素在50ml.ml的浓度下特异性识别D-半乳糖残留物时，子孢子的穿透被抑制。第二步，制备鸡抗艾美耳球虫顶端复合体的单抗，以便在入侵过程中获得更多的知识。通过将胸苷激酶(TK)缺陷的鸡骨髓瘤与子孢子抗原免疫的鸡的脾细胞融合，获得了许多稳定分泌检测艾美耳球虫子孢子顶端复合体的单抗的鸡杂交瘤细胞。其中一株单抗命名为6D-12-G10，识别20-21 kDa分子…的子孢子顶端复合体更多关于西方印迹的信息。免疫电子显微镜检查显示，mAb6D-12-G10对锥体抗原进行了染色。此外，mAb6D-12-G10在体外可抑制子孢子对CD8+T细胞的侵袭。下一步，利用激光共聚焦扫描显微镜和免疫印迹技术，以7种不同禽艾美耳球虫种的子孢子为材料，研究了鸡对艾美耳球虫(Ea)、布氏艾美耳球虫(Eb)、极大艾美耳球虫(Em)、米蒂斯艾美耳球虫(E.mitis)、艾美耳球虫(E.neatrix)、雷氏艾美耳球虫(E.preecox)和柔嫩艾美耳球虫(E.tenella)的种特异性和交叉反应性。本研究使用了5种不同的单抗，分别为8E-1、HE-4、8D-2、5D-11和8C-3。在免疫荧光检查中，这些单抗在子孢子顶端复合体上表现出相似的反应活性。其中8E-1单抗与所检测的所有艾美耳球虫种交叉反应，HE-4和8D-2单抗与EA和EB反应，5D-11单抗与EA和EM反应，8C-3单抗仅与EA反应。以7种艾美耳球虫的无孢子体为抗原进行Western印迹分析，所有单抗均识别出多条带，优势带约为32、43和260千道尔顿。目前宿主细胞表面特定糖基残基的研究结果表明，柔嫩艾美耳球虫子孢子表面的D-半乳糖残基和识别宿主细胞D-半乳糖的凝集素样受体是穿透的重要因素，这些单抗可能有助于更清楚地了解寄生虫入侵宿主细胞的结果。此外，我们还可以继续研究单抗识别的抗原与糖残留物之间的关系。开发鸡单抗的技术的可用性将极大地增强我们研究单个分子在附着、入侵和运动中的作用的能力。这些分子可能被证明是免疫和药物治疗球虫感染的重要和新的靶点。较少

项目成果

K.Sasai,et al.: "Characterization of a chicken monodonal antibody that recognizes the apical complex of Eimeria acervulinasporozoites and partially inhibits sporozoite in vasion of CD8+ Tlymphocytes in vitro." J.Parasitol.82. 641-644 (1996)

K.Sasai 等人：“一种鸡单克隆抗体的表征，该抗体可识别埃美耳球虫子孢子的顶端复合物，并在体外部分抑制子孢子侵入 CD8 T 淋巴细胞。”

K.Sasai,et al: "Cross-species and cross-strain reactivity of a chicken anti-conoid monocional antibody." J.Parasitol.(in press). (1998)

K.Sasai 等人：“鸡抗圆锥形单克隆抗体的跨物种和跨品系反应性。”

K.Sasai,et al: "Characterization of a chicken monoclonal antibody that recognizes the apical complex of Eimeria acervulina sporozoites and partialy inhibits sporozoite invasion of CD8+ T lymphocytes in vitro." J.Parasitol.82. 641-644 (1996)

K.Sasai 等人：“一种鸡单克隆抗体的表征，该抗体可识别艾美耳球虫子孢子的顶端复合物，并在体外部分抑制子孢子侵入 CD8 T 淋巴细胞。”

Sasai K., Y.Hanioka, H.S.Lillehoj, H.Matsuda, T., Fukata, E.Baba & A.Arakawa: "Cross-species and cross-strain reactivity of a chicken anti-conoid monoclonal antibody." Journal of Parasitology. (in press.). (1998)

Sasai K.、Y.Hanioka、H.S.Lillehoj、H.Matsuda、T.、Fukata、E.Baba

Sasaki K,Y.Hanioka,T.Fukata,E Baba & A.Arakawa et. al.: "Cross-species and cross-strain reactivity of a chicken anti-conoid monoclonal antibody." Journal of Parasitology. (in press). (1998)

佐佐木 K、Y.Hanioka、T.Fukata、E Baba