Bochsler P N, Yang Z, Murphy C L, Carroll R C
Department of Pathology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901, USA.
Vet Immunol Immunopathol. 1996 Jun 1;51(3-4):303-14. doi: 10.1016/0165-2427(95)05523-1.
Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56 degrees C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62 degrees C for 30 min, which compared favorably with data resulting from use of fetal bovine serum.
脂多糖结合蛋白（LBP）在将细菌衍生的脂多糖（LPS；内毒素）呈递给白细胞（如巨噬细胞和中性粒细胞）的过程中起着核心作用。LBP与LPS的相互作用意义重大，因为LBP-LPS复合物可促进白细胞和免疫系统的激活，进而导致一系列促炎细胞因子的分泌增加。采用一种改进的简化方法从血清中纯化牛LBP。方法包括使用Bio-Rex 70树脂进行离子交换色谱，随后对选定的离子交换级分（0.22 - 0.50 M NaCl）进行凝胶过滤色谱（Sephacryl S-200树脂）。对色谱分离得到的蛋白质进行银染聚丙烯酰胺凝胶的光密度扫描表明，最纯的级分中所得64kD蛋白（牛LBP）的纯度高达88.7%。测定牛LBP的等电点为6.8。通过蛋白质印迹分析以及N端氨基酸序列分析证实了该蛋白的身份，与已发表的兔、人及小鼠LBP序列数据进行了良好比较。通过在生物测定中使用纯化的牛LBP进一步证实了其身份，该生物测定表明LPS（1 ng ml(-1)）刺激的牛肺泡巨噬细胞的组织因子表达增强。在这些测定中，使用抗CD14单克隆抗体可抑制组织因子表达，这也与LBP介导的细胞激活一致。当牛LBP在56℃加热30分钟时，基于巨噬细胞的生物测定中其生物活性降低了50%。在62℃加热30分钟可完全破坏牛LBP的生物活性，这与使用胎牛血清得到的数据相比效果良好。
