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【目的】建立分离、纯化分枝杆菌脂聚糖的方法,初步比较分析不同菌株来源的脂阿拉伯甘露聚糖(Lipoarabinomannan,LAM)和脂甘露聚糖(Lipomannan,LM)的结构差异及研究脂聚糖刺激对巨噬细胞环氧合酶-2(Cyclooxygenase-2,COX-2)蛋白表达的影响。【方法】应用Triton X-114液相法提取脂聚糖,电洗脱法分离纯化,基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF/TOF-MS)进行分子量鉴定;基于特异性识别非还原性末端α-D-甘露糖基的刀豆球蛋白(Concanavalin A,Con A)分析新诺分枝杆菌JDM601、结核分枝杆菌H37Rv标准株和耻垢分枝杆菌mc2155脂聚糖的结构差异;进一步用Western blot检测脂聚糖刺激的RAW 264.7巨噬细胞COX-2蛋白的表达。【结果】通过电洗脱法成功纯化出3种菌株脂聚糖;MALDI-TOF/TOF-MS鉴定发现,分子量从小到大依次为新诺分枝杆菌JDM601、耻垢分枝杆菌mc2155和结核分枝杆菌H37Rv来源的脂聚糖。Western blot显示,Con A能与结核分枝杆菌H37Rv标准株来源的LAM相互作用,而不能与新诺分枝杆菌JDM601和耻垢分枝杆菌来源的LAM相互作用;并且发现Con A与新诺分枝杆菌JDM601来源的LM有很强的反应,然而与其余两种来源的LM反应很弱。3种菌株来源的脂聚糖均能刺激RAW 264.7巨噬细胞COX-2蛋白的表达。【结论】首次成功对来源于中国临床分枝杆菌分离株的脂聚糖进行了分离纯化,初步探讨了不同菌株来源分枝杆菌脂聚糖的结构差异,并表明LAM和LM均能刺激巨噬细胞诱导COX-2蛋白的表达,为进一步研究其对宿主的毒力和免疫机制奠定了基础。
【Objective】 To establish a method for the isolation and purification of mycobacterium lipopolysaccharide and to analyze the structural differences of lipoarabinomannan (LAM) and lipomannan (LM) Effects of sugar stimulation on the expression of cyclooxygenase-2 (COX-2) protein in macrophages. 【Method】 Lipopolysaccharide was extracted by liquid phase method using Triton X-114, and separated by electroelution. The molecular weight was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF / MS) Reducing terminal α-D-mannosyl Concanavalin A (Con A) The structural differences between Mycobacterium tuberculosis JDM601, Mycobacterium tuberculosis H37Rv standard strain and Mycobacterium smegmatis mc2155 lipopolysaccharide were analyzed The expression of COX-2 protein in RAW 264.7 macrophages stimulated by lipopolysaccharide was further detected by Western blot. 【Result】 Three strains of lipopolysaccharide were successfully purified by electroelution method. The identification of MALDI-TOF / TOF-MS showed that the order of the molecular weights was Mycobacterium denatus JDM601, Mycobacterium smegmatis mc2155 and tuberculosis Mycobacterium H37Rv-derived lipopolysaccharide. Western blot showed that Con A interacted with LAM derived from Mycobacterium tuberculosis H37Rv standard strain, but not with Lactobacillus casei JDM601 and Mycobacterium smegmatis, and found that Con A and Newnot Mycobacterium JDM601-derived LM has a strong response, however, with the other two sources of LM reaction is very weak. All three strains of lipopolysaccharide can stimulate the expression of COX-2 protein in RAW 264.7 macrophages. 【Conclusion】 The first successful isolation and purification of lipopolysaccharide from clinical Mycobacterium isolates in China, preliminary study of different strains of mycobacterium lipolytica structural differences, and that both LAM and LM can stimulate macrophages Cells induce the expression of COX-2 protein, which lays the foundation for further study of its virulence and immune mechanism on the host.