Li D M, Liu Q Y, Zhao F, Hu Y, Xiao D, Gu Y X, Song X P, Zhang J Z
Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
Genet Mol Res. 2011 Aug 26;10(3):1789-818. doi: 10.4238/vol10-3gmr1153.
Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.
汉赛巴尔通体是一种可导致人类猫抓病和血管增殖性疾病的感染因子，是一种苛求性兼性细胞内病原体。汉赛巴尔通体的外膜蛋白是在宿主细胞相互作用中起主要作用的关键分子。我们使用离子去污剂N-月桂酰肌氨酸钠盐和碳酸钠分离汉赛巴尔通体的外膜蛋白，通过二维（2-D）凝胶电泳进行纯化，并使用质谱法进行蛋白质鉴定。用含有ASB-14和两性离子去污剂3-10的缓冲液处理可增加汉赛巴尔通体蛋白的溶解性，尤其是具有碱性pI的蛋白。从肌氨酸不溶性外膜部分检测到368个斑点；从176个斑点中鉴定出94种不同的蛋白质。在碳酸盐孵育的外膜部分中，计算出471个斑点并鉴定出259个斑点，其中包括139个蛋白质条目。与经过碳酸盐孵育的样品中的9种外膜蛋白相比，肌氨酸不溶性外膜部分中有6种外膜蛋白。我们通过预测其结构域和三级结构，使用生物信息学分析鉴定了44种外膜蛋白，并记录了潜在的毒力因子。我们使用两种不同的提取方法建立了汉赛巴尔通体外膜亚蛋白质组的二维参考图谱，这两种方法部分相互补充。碳酸钠提取比月桂酰肌氨酸钠盐提取能更好地分离低丰度和碱性蛋白，月桂酰肌氨酸钠盐提取则富集了高丰度的孔蛋白。
