Wise K S, Kim M F
Department of Microbiology, School of Medicine, University of Missouri-Columbia 65212.
J Bacteriol. 1987 Dec;169(12):5546-55. doi: 10.1128/jb.169.12.5546-5555.1987.
Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species.
猪肺炎支原体的表面蛋白抗原通过直接抗体-表面结合或用一系列单克隆抗体（MAb）对表面125I标记蛋白进行放射免疫沉淀来鉴定。表面蛋白p70、p65、p50和p44在Triton X-114（TX-114）相分离过程中通过选择性分配到疏水相而被证明是完整的膜成分，而p41则被同时鉴定为仅分配到水相的表面蛋白。对用[35S]甲硫氨酸、14C-氨基酸或[3H]棕榈酸标记的细胞的TX-114相蛋白进行放射免疫沉淀表明，蛋白p65、p50和p44含量丰富，并且（与另一种疏水蛋白p60一起）被脂质选择性标记。通过对脱脂蛋白进行酸性甲醇解后用高效液相色谱法鉴定[3H]棕榈酸甲酯，确定了共价脂质连接。另一种未鉴定的甲醇解产物表明棕榈酸转化为也与这些蛋白连接的另一种脂质形式。对标记蛋白进行碱性羟胺处理表明脂质通过酰胺或稳定的O-连接酯键连接。用接种了全菌的猪的抗血清对TX-114相蛋白进行免疫印迹分析，结果表明蛋白p65、p50和p44在天然宿主中具有高度免疫原性。这些蛋白在抗原性和结构上不相关，因为针对单个凝胶纯化蛋白的超免疫小鼠抗体是单特异性的，并给出了不同的蛋白水解表位图谱。一种针对p70的单克隆抗体（在J株，ATCC 25934中定义）揭示了猪肺炎支原体一种表面抗原的种内大小变异体，该抗体识别VPP11株（ATCC 25617）上一个更大的p73成分。此外，针对J株内部水相蛋白p82的单克隆抗体未能与VPP11株中的类似抗原结合。这些研究表明，一组高度受限的不同的、脂质修饰的疏水膜蛋白是猪肺炎支原体的主要表面抗原，并且该物种内存在表面抗原的结构变异体。
