分枝杆菌多糖的制备及对小鼠脾淋巴细胞增殖的影响

Preparation of Mycobacterium polysaccharides and their effect on lymphocyte proliferation

目的：制备分枝杆菌多糖并考察其对小鼠淋巴细胞增殖的影响。方法：用苏通培养基培养分枝杆菌,菌体脱脂后,用5种方法提取,比较多糖得率,并用正交试验优化超声提取工艺,再用稀碱提取残渣,粗多糖经Sevag法除蛋白、DEAE-Sepharose Fast Flow层析柱纯化后得到分枝杆菌多糖;用MTT法检测分枝杆菌多糖对小鼠脾淋巴细胞增殖的影响。结果：超声后热水提取多糖得率最高,超声提取的优化工艺是提取时间40 min,固液比为1∶150,超声功率600 W;单因素试验优化的Sevag法除蛋白条件为体积比1∶5,萃取时间为20 min,萃取6次;提取得到的4种多糖组分在6.25~50 mg.L-1范围内均能显著促进脾淋巴细胞的增殖。结论：用本实验工艺成功制备了分枝杆菌多糖,制备物具有刺激小鼠脾淋巴细胞增殖的作用。

Objective： To prepare Mycobacterium polysaccharides（MPS） and determine their effect on lymphocyte proliferation in mice.Methods： Mycobacterium was cultivated in Sauton＇s medium,and 5 methods were used to extract polysaccharides,and the extraction rates were compared.The ultrasonic extraction was optimized by an orthogonal design.Hot-water extraction residue was extracted in alkaline solution.Crude polysaccharides were purified by deproteinization and DEAE-sepharose Fast Flow chromatography.The effect of polysaccharides on mouse splenic lymphocytes was analyzed by MTT method.Results： The highest polysaccharide yield was obtained by ultrasonic and followed hot-water extraction;the best ultrasonic extraction condition defined by the orthogonal design was extracting 40 min with 150 times solvent and 600 W power.The best deproteiniation condition was extracting 6 times with 1/5 Sevag reagent,20 min for every time.All the obtained 4 kinds of mycobacterium polysaccharides apparently stimulated the proliferation of mouse splenic lymphocytes at concentrations of 6.25~50 mg·L-1.Conclusion： Mycobacterium polysaccharides are successfully prepared by this method and they can stimulate the proliferation of mouse splenic lymphocytes.