Meens Jochen, Selke Martin, Gerlach Gerald-F
Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation Hannover, Germany.
Vet Microbiol. 2006 Aug 25;116(1-3):85-95. doi: 10.1016/j.vetmic.2006.03.011. Epub 2006 May 2.
Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized, species-specific M. hyopneumoniae antigens. As a first step to solve these problems membrane and membrane-associated proteins were enriched from M. hyopneumoniae cells by Triton X-114 fractionation and further analyzed by 2D gel electrophoresis and Western blot analyses using convalescent sera. Two previously unknown immunogenic proteins were identified by quadrupole time-of-flight mass spectrometry and database analyses as the conserved putative lipoproteins, Mhp378 and Mhp651. Both proteins were expressed as recombinant GST fusion proteins and reacted with sera from convalescent pigs. Coated as solid-phase antigen, Mhp651 showed a distinct cross-reaction only with Mycoplasma flocculare specific rabbit hyperimmune serum, whereas Mhp378 was only recognized by the positive control serum directed against M. hyopneumoniae, thereby indicating its species specificity.
猪肺炎支原体是猪地方流行性肺炎的病原体，是全球养猪业中的一种重要病原体。由于缺乏已知且特征明确的猪肺炎支原体种特异性抗原，对其致病机制以及当前血清学检测方法和新型重组亚单位疫苗研发的研究受到了阻碍。作为解决这些问题的第一步，通过Triton X-114分级分离从猪肺炎支原体细胞中富集膜及膜相关蛋白，并使用恢复期血清通过二维凝胶电泳和蛋白质免疫印迹分析进行进一步分析。通过四极杆飞行时间质谱和数据库分析鉴定出两种先前未知的免疫原性蛋白为保守的假定脂蛋白，即Mhp378和Mhp651。这两种蛋白均表达为重组GST融合蛋白，并与恢复期猪的血清发生反应。作为固相抗原包被时，Mhp651仅与絮状支原体特异性兔超免疫血清显示出明显的交叉反应，而Mhp378仅被针对猪肺炎支原体的阳性对照血清识别，从而表明其种特异性。
