Cloeckaert A, Grépinet O, Salih-Alj Debbarh H, Zygmunt M S
Laboratoire de Pathologie infectieuse et Immunologie, Institut National de la Recherche Agronomique, Nouzilly, France.
Res Microbiol. 1996 Mar-Apr;147(3):145-57. doi: 10.1016/0923-2508(96)80214-2.
The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B. ovis and cloned in multiple copy plasmids enabling expression under the control of the Plac promoter. Monoclonal antibodies (mAb) obtained by immunizing mice with B. melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B. melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B. melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK. The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes. These mAbs were further used to study overproduction of the DnaK protein. B. melitensis DnaK overproduction in E. coli resulted in a defect in cell septation and formation of cell filaments. Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E. coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein. These results indicated that overproduction of the B. melitensis DnaK protein in E. coli had similar physiological consequences as that of E. coli overproduced in E. coli. The DnaK protein localization in B. melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy. Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm. However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B. melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.
使用根据绵羊布鲁氏菌已发表序列设计的引物，通过聚合酶链反应扩增了羊种布鲁氏菌dnaK基因，并将其克隆到多拷贝质粒中，使其能够在Plac启动子的控制下表达。用羊种布鲁氏菌B115细胞壁（CW）组分免疫小鼠或用强毒株羊种布鲁氏菌H38感染小鼠获得的单克隆抗体（mAb），在羊种布鲁氏菌CW组分的免疫印迹中识别出一条73 kDa的条带，该抗体与克隆的dnaK基因产物发生反应，因此被证明对热休克蛋白DnaK具有特异性。抗Dnak蛋白单克隆抗体不与大肠杆菌对照细胞或细胞裂解物发生反应，因此可能对布鲁氏菌DnaK蛋白表位具有特异性。这些单克隆抗体进一步用于研究DnaK蛋白的过量表达。羊种布鲁氏菌DnaK在大肠杆菌中的过量表达导致细胞分裂缺陷和细胞丝的形成。用单克隆抗体进行免疫金标记和电子显微镜观察发现，DnaK蛋白存在于大肠杆菌细胞内外，这可能是由于过量产生的DnaK蛋白毒性导致细胞裂解所致。这些结果表明，羊种布鲁氏菌DnaK蛋白在大肠杆菌中的过量表达与大肠杆菌在大肠杆菌中过量表达具有相似的生理后果。免疫电子显微镜显示，羊种布鲁氏菌细胞中DnaK蛋白主要位于细胞质中。对这些细胞进行热休克处理后，单克隆抗体的结合增加，细胞质中的标记增多。然而，在亚细胞组分中，DnaK蛋白主要存在于羊种布鲁氏菌的细胞膜组分中，这可能是由于DnaK蛋白与膜蛋白相互作用的结果。
