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

已知入侵原生动物识别宿主细胞表面的特异性糖残基，并利用顶端复合物侵入细胞。在这一系列研究的第一步，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进行了研究。当用50 ml. ml的花生凝集素预处理时，子孢子的穿透被抑制，所述花生凝集素特异性地识别D-半乳糖残基。第二步，制备抗艾美耳球虫顶端复合体的单克隆抗体，为进一步了解艾美耳球虫的入侵过程奠定基础。将胸苷激酶（TK）缺陷型鸡骨髓瘤细胞与子孢子抗原免疫鸡脾细胞融合，获得了稳定分泌单克隆抗体（mAb）的杂交瘤细胞。其中一种单克隆抗体，命名为6D-12-G10，识别分子量为20 - 21 kDa的子孢子的顶端复合物， ...更多信息 蛋白质印迹法。免疫电镜检查显示，mAb 6D-12-G10染色锥状抗原。此外，mAb 6D-12-G10在体外抑制子孢子侵入CD8 + T细胞。采用激光共聚焦扫描显微镜和Western blotting技术，检测了鸡源艾美耳球虫（E.acervulina，EA）、布氏艾美耳球虫（E.brunetti，EB）、巨型艾美耳球虫（E.maxima，EM）、缓症艾美耳球虫（E.mitis）、毒害艾美耳球虫（E.necatrix）、早熟艾美耳球虫（E.praecox）和柔嫩艾美耳球虫（E.tenella）7种不同种类的子孢子对鸡源艾美耳球虫（E.acervulina，EA）的种特异性和交叉反应性。本研究中使用了五种不同的mAb，命名为8E-1、HE-4、8D-2、5D-11和8C-3。在免疫荧光检测中，这些单克隆抗体对子孢子的顶端复合物显示出相似的反应性。其中8E-1单抗可与所检测的所有艾美耳球虫交叉反应，HE-4和8D-2单抗可与EA和EB反应，5D-11单抗可与EA和EM反应，8C-3单抗仅与EA反应。mAb的主要条带具有约32、43和260千道尔顿的分子量。宿主细胞表面特异性糖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其穿透的重要因素，这些单克隆抗体可能有助于更清楚地检测寄生虫侵入宿主细胞的结果。同时，也为进一步研究单克隆抗体识别的抗原与糖残基的关系奠定了基础。开发鸡单克隆抗体的技术的可用性将大大提高我们研究参与附着、侵袭和运动的单个分子的作用的能力。这些分子可能被证明是重要的和新的目标免疫和药物治疗抗球虫感染。少

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