Frey J, Deillon J B, Gygi D, Nicolet J
Institute for Veterinary Bacteriology, University of Berne, Switzerland.
Vet Microbiol. 1991 Aug 15;28(3):303-12. doi: 10.1016/0378-1135(91)90085-t.
The secreted hemolytic activity produced by Actinobacillus pleuropneumoniae serotype 2 reference strain is thermolabile, inactivated by proteinase K and requires Ca2+ as cofactor for its hemolytic activity. Purification of the hemolytic activity resulted in a fraction containing two proteins, one of 105 kDa and one of 125 kDa. These two proteins could be further separated by preparative SDS polyacrylamide gel electrophoresis. This purification step, resulted in loss of the hemolytic activity. Polyclonal antibodies were made against each of these proteins in rabbits. Neutralization experiments showed that antibodies made against the 105 kDa protein could neutralize the hemolytic activity produced by A. pleuropneumoniae serotype 2, while antibodies made against the 125 kDa protein were unable to neutralize the hemolytic activity. The 105 kDa protein therefore, is the hemolysin of A. pleuropneumoniae serotype 2, known as HlyII. This protein is closely related immunologically to the hemolysin I (HlyI) from A. pleuropneumoniae serotype 1. DNA::DNA hybridization experiments performed by the Southern blot method using the cloned structural gene of HlyI from A. pleuropneumoniae serotype 1 demonstrate that the structural genes of the two hemolysins (hlyIA and hlyIIA) are different and show at least 30% heterology. This confirms that HlyI and HlyII are two different proteins, although they have a very similar molecular weight and show strong immunological cross reactions.
胸膜肺炎放线杆菌2型参考菌株产生的分泌型溶血活性具有热不稳定性，可被蛋白酶K灭活，且其溶血活性需要Ca2+作为辅助因子。对溶血活性进行纯化后得到一个含有两种蛋白质的组分，一种为105 kDa，另一种为125 kDa。这两种蛋白质可通过制备性SDS聚丙烯酰胺凝胶电泳进一步分离。这一纯化步骤导致溶血活性丧失。用这些蛋白质分别在兔体内制备了多克隆抗体。中和实验表明，针对105 kDa蛋白质制备的抗体能够中和胸膜肺炎放线杆菌2型产生的溶血活性，而针对125 kDa蛋白质制备的抗体则无法中和溶血活性。因此，105 kDa蛋白质是胸膜肺炎放线杆菌2型的溶血素，称为HlyII。该蛋白质在免疫学上与胸膜肺炎放线杆菌1型的溶血素I（HlyI）密切相关。使用胸膜肺炎放线杆菌1型HlyI的克隆结构基因通过Southern印迹法进行的DNA::DNA杂交实验表明，这两种溶血素的结构基因（hlyIA和hlyIIA）不同，且显示出至少30%的异源性。这证实了HlyI和HlyII是两种不同的蛋白质，尽管它们的分子量非常相似且显示出强烈的免疫交叉反应。
