Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae.
Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae.
复制标题
恶丝虫感染性幼虫主要表面抗原的生化和免疫学特征。
作者：
M. Philipp;T. B. Davis
用生化和免疫学方法研究了金丝虫三期幼虫35kD的主要表面抗原。活体幼虫用碘-根、碘磺酸、乳过氧化物酶-葡萄糖氧化酶和Bolton-Hunter试剂进行碘化。用前三种方法标记的幼虫洗涤剂提取物经一维SDS-PAGE分析，显示出一个主要的35kD组分和一些约6kD的较小组分。相反，用Bolton-Hunter试剂标记的幼虫提取物在凝胶上显示多条带。35kD分子暴露在幼虫表面，在活体放射性标记幼虫上可被胰酶蛋白分解。双向凝胶电泳法将35kD的条带分成两个组分：一个大的等电点为3.8，一个小的等电点为7.3。较低的M.W.将谱带分解为12个组分，等电点在3.5~8.0之间。在所有这些表面分子中，唯一具有抗原性的是35kD组分。它可以用携带隐匿性实验性吸虫性感染的狗的血清或用这种寄生虫的辐照第三期幼虫免疫的狗的血清免疫沉淀。同样，用正常未照射的幼虫反复免疫的兔血清也沉淀出35kD的抗原。然而，这些血清中没有一种含有可检测到的表面标记的低分子量抗体。分子。用吸虫成虫和微丝虫免疫的兔血清沉淀出35kD的抗原，因此不是阶段特异性的。相比之下，实验感染犬弓形虫和犬钩虫或狭头钩虫的狗的血清不包含针对35kD抗原的抗体，但与许多其他吸虫成虫和微丝虫抗原发生交叉反应。因此，这种分子可能是物种特有的。没有发现35kD分子糖基化的证据：它不与花生、小麦胚芽、小扁豆或黑曲霉凝集素结合，经内切糖苷酶-F或温和碱性溶液处理后，其凝胶迁移率没有改变。
A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.