Singh B N, BonDurant R H, Campero C M, Corbeil L B
Department of Biochemistry and Molecular Biology, SUNY UpState Medical University, Syracuse, New York 13210, USA.
J Parasitol. 2001 Aug;87(4):770-7. doi: 10.1645/0022-3395(2001)087[0770:IABAOG]2.0.CO;2.
Immunoaffinity-purified TF1.17 adhesin antigen was compared biochemically and antigenically to Tritrichomonas foetus (TF) lipophosphoglycan (LPG) and a soluble glycosylated antigen (SGA) released from T. foetus and implicated in pathogenesis and immunity. The monoclonal antibodies (Mabs TF1.15 and TF1.17) specific for a glycosylated TF1.17 antigen were previously shown to prevent adhesion of the T. foetus parasites to bovine vaginal epithelial cells and to mediate killing by bovine complement. SGA was isolated from T. foetus-conditioned buffer and purified by octyl-Sepharose hydrophobic column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SGA showed a major SGA1 component (approximately 190 kDa) and a minor SGA2 component (50-70 kDa), which migrated close to TF-LPG and TF1.17. The carbohydrate and lipid compositional analyses of affinity-purified TF1.17 and SGA2 by high-performance liquid chromatography (HPLC) and gas-liquid chromatography revealed the presence of monosaccharides and fatty acids as found in TF-LPG. All antigens contained terminal fucose as determined by alpha-fucosidase digestion followed by HPLC. ELISA and western blots were used to further characterize these glycosylated antigens and to analyze their relationships. The Mabs TF1.15 and TF1.17 reacted very strongly to TF-LPG and SGA2. as well as TF1.17 antigen, indicating that these molecules share common epitopes. These Mabs did not react with the SGA1 component either in ELISA and western blot analyses. Also, the monosaccharide composition of SGA1 was very different from the other three antigen, suggesting SGA1 was different from LPG, SGA2 and TF1.17. Although LPG reacted with Mabs to native TF1.17 antigen, LPG did not induce an immune response in cattle with the same route and adjuvant used to produce strong antibody responses to the native antigen. The latter response suggests that the tightly bound peptide present in the immunoaffinity-purified antigen is necessary for induction of a response to (an) epitope(s) in TF-LPG and TF1.17. Furthermore, vaginal fluid from T. foetus-infected heifers and serum from a cow with a T. foetus-associated pyometra recognized both TF1.17 and TF-LPG in western blots. These results suggest that T. foetus LPG and SGA2 are related to TF1.17 antigen, which was previously shown to play an important role in the pathogenesis and host response in bovine trichomoniasis.
将免疫亲和纯化的TF1.17黏附素抗原与胎儿三毛滴虫（TF）脂磷壁酸聚糖（LPG）以及从胎儿三毛滴虫释放的一种可溶性糖基化抗原（SGA）进行生化和抗原性比较，SGA与发病机制及免疫相关。先前已证明，对糖基化TF1.17抗原有特异性的单克隆抗体（Mabs TF1.15和TF1.17）可阻止胎儿三毛滴虫寄生虫黏附于牛阴道上皮细胞，并介导牛补体的杀伤作用。SGA从胎儿三毛滴虫条件缓冲液中分离出来，并通过辛基琼脂糖疏水柱色谱法进行纯化。SGA的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示，主要的SGA1成分（约190 kDa）和次要的SGA2成分（50 - 70 kDa），其迁移位置接近TF-LPG和TF1.17。通过高效液相色谱（HPLC）和气-液色谱对亲和纯化的TF1.17和SGA2进行碳水化合物和脂质成分分析，结果显示存在TF-LPG中发现的单糖和脂肪酸。通过α-岩藻糖苷酶消化后再进行HPLC测定，所有抗原均含有末端岩藻糖。采用酶联免疫吸附测定（ELISA）和蛋白质印迹法进一步表征这些糖基化抗原，并分析它们之间的关系。Mabs TF1.15和TF1.17与TF-LPG、SGA2以及TF1.17抗原反应非常强烈，表明这些分子具有共同的表位。在ELISA和蛋白质印迹分析中，这些单克隆抗体均不与SGA1成分反应。此外，SGA1的单糖组成与其他三种抗原非常不同，这表明SGA1与LPG、SGA2和TF1.17不同。尽管LPG与针对天然TF1.17抗原的单克隆抗体发生反应，但在用于对天然抗原产生强烈抗体反应的相同途径和佐剂条件下，LPG并未在牛体内诱导免疫反应。后一种反应表明，免疫亲和纯化抗原中存在的紧密结合肽对于诱导对TF-LPG和TF1.17中表位的反应是必需的。此外，来自感染胎儿三毛滴虫的小母牛的阴道分泌物以及患有与胎儿三毛滴虫相关的子宫积脓的母牛的血清在蛋白质印迹中均能识别TF1.17和TF-LPG。这些结果表明，胎儿三毛滴虫LPG和SGA2与TF1.17抗原相关，先前已证明TF1.17抗原在牛滴虫病的发病机制和宿主反应中起重要作用。
