兔源内毒素结合蛋白的提取纯化及体外生物学活性研究

A study of the extraction and purification of rabbit lipopolysaccharide-binding protein and its biological function in vitro

目的 从烧伤合并内毒素血症损伤兔的血清中分离纯化出内毒素结合蛋白 (Lipopolysaccharide BindingProteinLBP) ,以研究LBP在烧伤复合内毒素血症中的作用。方法 兔血清中蛋白质通过二步离子交换层析 (Bio Rex 70Resin、Mono Q)及凝胶过滤来分离纯化 ,并经流式细胞分析实验、绵羊红细胞凝聚实验、氨基末端氨基酸残基测序等方法鉴定 ,再将LBP作用于体外培养的人单核细胞 (U93 7) ,观测其分泌TNFα等细胞因子的状况。结果 获得一种分子量为 60× 10 3蛋白质 ,其N端 10个氨基酸序列为TNPGLITRIT ,与NCBI蛋白质库中有关兔的氨基酸序列进行比较 ,发现其与LBP的序列相同 ,它能促进脂多糖 (LipopolysaccharideLPS)与外周血单核细胞 (PBMC)结合 ,并能促进U93 7在极低浓度的LPS用下分泌TNFα。结论 从血清中分离纯化带有生物活性、全长LBP是可行的 ,LBP能促进极低浓度的LPS与单核细胞结合 ,介导炎性因子的分泌 ,从而促进炎症反应的发生。

Objective To purify and extract the lipopolysaccharide binding protein from the serum in rabbits inflicted with injury due to burn complicated with endotoxemia so as to study the principal biological function of the protein. Methods The protein of the rabbit serum was purified and extracted by two step ion exchange chromatography (Bio Rex 70 Resin, Mono Q) and gel chromatography, and was determined with fluorescence activated cell sorter(FACS) experiment, agglomerate experiment of sheep erythrocyte and NH 2 amino acid sequence determination. At last LBP was added to the culture of human monocyte (U937) to observe the changes of the tumor necrosis factor alpha (TNFα) in the culture. Results A kind of protein with molecular weight being 60kDa was acquired from the serum. Its NH 2 terminal amino acid sequence was TNPGLITRIT. After comparison with the rabbit amino acid sequence bank of NCBI (National Center for Biotechnology Information), it was proved to be the same sequence as that of LBP. The protein could promote the binding of LPS (lipopolysaccharide) with peripheral blood mononuclear cells(PBMC) and accelerate the secretion of TNFα by U937 with low molarity of LPS. Conclusion It is feasible to acquire complete functional LBP from serum. LBP can promote the inflammatory response by the acceleration of the binding of LPS at low concentration with monocyte and induction of the secretion of inflammatory factor.