一种新型兔源内毒素结合蛋白的分离纯化及体外生物活性的初步研究

The purification and isolation of a new type of rabbit-origin lipopolysaccharide binding protein and the study of its biological function in vitro

目的 分离纯化一种新型的内毒素结合蛋白 ,并初步观察其生物学特性。 方法 建立兔烧伤合并内毒素血症模型 ,采其血清 ,经二步离子交换层析及凝胶过滤分离纯化 ,并经流式细胞仪分析、绵羊红细胞凝聚试验、氨基末端氨基酸残基测序等方法鉴定。将所获蛋白质作用于体外培养的人单核细胞 (U937) ,观测其分泌TNFα等细胞因子的状况。 结果 所获蛋白质的分子量为 48kDa( 48× 10 3) ,其N端 10个氨基酸序列为GSQGTFTSEE ,与美国国立图书馆蛋白质库中有关兔的氨基酸序列进行比较 ,未发现相同序列 ,暂命名为P48。它具有与内毒素结合蛋白相似的功能 ,能促进极低浓度的脂多糖 (lipopolysaccharide ,LPS)与外周血单核细胞 (PBMC)结合 ,并促进U937分泌TNFα。 结论 从兔血清中能够分离纯化出带有生物学活性的新型内毒素结合蛋白P48,它具有与脂多糖结合蛋白 (lipoplysaccharidebindingprotein ,LBP)相似的生物学活性 ,并可促进炎症反应的发生。

Objective To isolate and purify a new type of lipopolysaccharide binding protein (LBP) from burn rabbit serum, and to investigate its biological functions. Methods Rabbits subjected to burn injury and endotoxemia were employed. The serum from the rabbits was purified by two-steps of ion-exchange chromatography (Bio-Rex 70 Resin, Mono-Q) and gel chromatography. Furthermore, the serum was identified by flow cytometry analysis, agglomeration test with sheep erythrocyte, and amino end amino acid residue sequencing. The obtained protein was applied to cultured human monocytes (U937), and the cytokine secretion such as TNFα from the U937 was observed. Results The molecular weight of the harvested protein was 48kDa, and the 10 amino acid sequence at N end was arranged as GSQGTFTSEE, which was different to the amino acid sequence in NCBI protein bank and was so named P48. P48 possessed similar function to that of LBP and could promote the binding of LPS in a very low concentration with peripheral blood monocytes(PBMC), and also promote the TNFα secretion from U937. Conclusion P48, a new type of LBP, could be isolated and purified from the burn rabbit serum. P48 possesed similar biological activities to that of LBP and could promote the process of inflammatory reaction. [