Gerlach G F, Anderson C, Potter A A, Klashinsky S, Willson P J
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
Infect Immun. 1992 Mar;60(3):892-8. doi: 10.1128/iai.60.3.892-898.1992.
An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.
从胸膜肺炎放线杆菌7型构建了一个表达文库。通过用猪恢复期血清进行免疫筛选，鉴定出表达重组蛋白的大肠杆菌转化体。鉴定出一个表达聚集形式的60 kDa蛋白（60K蛋白）的转化体。用重组蛋白免疫的血清仅在铁限制生长后的胸膜肺炎放线杆菌野生型中识别出一种电泳迁移率无法区分的多肽。将重组蛋白印迹到硝酸纤维素膜上后，它能结合转铁蛋白。使用竞争性酶联免疫吸附测定（ELISA），确定了这种结合对铁饱和猪转铁蛋白氨基末端一半的特异性。此外，通过血红素-琼脂糖色谱法评估，60K野生型蛋白能结合血红素。在竞争性ELISA中，血红素可抑制重组蛋白与转铁蛋白的结合，而血红蛋白和合成铁螯合剂则不能。对其他几种胸膜肺炎放线杆菌菌株的Southern印迹分析表明，在7型的8个分离株中的8个以及2型、3型和4型的一些分离株中存在高度同源序列。
