Rapp V J, Ross R F
Infect Immun. 1986 Dec;54(3):751-60. doi: 10.1128/iai.54.3.751-760.1986.
Sera from pigs infected with Haemophilus (Actinobacillus) pleuropneumoniae were tested for antibodies to outer membrane proteins (OMPs) of the organism by immunoblotting. Convalescent sera were produced in naturally born, colostrum-fed pigs and in cesarean-derived, colostrum-deprived pigs given H. pleuropneumoniae serotype 5 intranasally twice at 5-week intervals. Sera, collected at weekly intervals, were reacted with Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumoniae which had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Antibodies were detected to OMPs with an apparent molecular weight of 16,500 (16.5K OMP); to 29K, 38.5K, 43.5K, 45K, 49.5K, and 66.5K OMPs; and to several high-molecular-weight (greater than or equal to 94,000) OMPs, but not to the major 42K OMP. Antibodies to the heat-modifiable OMP (29K/43.5K) and the 38.5K OMP were detected in sera from noninfected pigs. Antibodies were also detected to two broad 54,000- and 95,000-molecular-weight bands which did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, and were eliminated by oxidation with sodium metaperiodate. This indicates that the 54,000- and 95,000-molecular-weight bands represent polysaccharide, possibly capsular or lipopolysaccharide immunogens. Adsorption of sera with cells from the homologous serotype 5 strain removed antibodies to the 45K, 49.5K, 66.5K, and greater than or equal to 94K OMPs and to the two polysaccharide bands, indicating that these antibodies were directed primarily to surface-exposed epitopes. When tested with OMP preparations from other serotype 5 strains, heterogeneity was apparent, both in the reactions with OMPs and with the polysaccharide bands. Silver staining of proteinase K-treated, whole-cell lysates from serotype 5 strains also indicated variable expression of the polysaccharide bands. Sera also reacted with OMPs from H. pleuropneumoniae serotypes 1 and 7; however, several OMPs and the lipopolysaccharide or polysaccharide determinants of these serotypes appeared to be type specific.
通过免疫印迹法检测感染胸膜肺炎放线杆菌（以前称胸膜肺炎嗜血杆菌）的猪血清中针对该菌外膜蛋白（OMPs）的抗体。康复血清采自自然分娩、初乳喂养的猪以及剖腹产、未接触初乳的猪，这些猪在5周龄时每隔5周经鼻内接种两次5型胸膜肺炎放线杆菌。每周采集血清，与经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离并电转移至硝酸纤维素膜上的胸膜肺炎放线杆菌的 Sarkosyl 不溶性、富含OMP的制剂反应。检测到针对表观分子量为16,500（16.5K OMP）的OMPs的抗体；针对29K、38.5K、43.5K、45K、49.5K和66.5K OMPs的抗体；以及针对几种高分子量（大于或等于94,000）OMPs的抗体，但未检测到针对主要的42K OMP的抗体。在未感染猪的血清中检测到针对热可修饰OMP（29K/43.5K）和38.5K OMP的抗体。还检测到针对两条宽的分子量为54,000和95,000的条带的抗体，这些条带用考马斯亮蓝不染色，用硝酸银染色，能抵抗蛋白酶K消化，并且经偏高碘酸钠氧化后消失。这表明分子量为54,000和95,000的条带代表多糖，可能是荚膜或脂多糖免疫原。用同源5型菌株的细胞吸附血清可去除针对45K、49.5K、66.5K和大于或等于94K OMPs以及两条多糖条带的抗体，这表明这些抗体主要针对表面暴露的表位。当用来自其他5型菌株的OMP制剂检测时，无论是与OMPs还是与多糖条带的反应中，异质性都很明显。5型菌株经蛋白酶K处理的全细胞裂解物的银染色也表明多糖条带的表达存在差异。血清也与1型和7型胸膜肺炎放线杆菌的OMPs反应；然而，这些血清型的几种OMPs以及脂多糖或多糖决定簇似乎具有型特异性。
