海胆黄多糖的分离、纯化及免疫活性测定

Extraction, purification and immunological activity assay of a polysaccharide from eggs of sea urchin Strongylocentrotus nudus

目的从光棘球海胆中分离纯化海胆黄多糖(polysaccharidefromtheeggsofStrongylocentrotusnu-dus,简称SEP),确定其纯度和分子量,并现察其免疫活性。方法海胆黄先经丙酮脱脂,根据正交实验和活性分析确定最佳热水提取条件,然后热水提取、去蛋白、醇沉得海胆黄粗多糖。粗多糖经超滤、DEAESepharoseFastFlow及SephacrylS-400柱层析纯化得多糖精品SEP。经高效液相色谱、聚丙烯酰胺凝胶电泳及纸层析鉴定其纯度。高效凝胶渗透色谱法(HPGPC)测定其分子量。体外脾淋巴细胞增殖实验测定其免疫活性。结果从海胆黄中分离纯化得到的均一多糖组分SEP,经检测其分子量为1950KD左右。脾淋巴细胞增殖实验表明SEP可显著促进脾淋巴细胞的增殖。结论从海胆黄中分离纯化得到的均一多糖组分SEP具有较强的体外免疫活性。

Objective To extract and purify a polysaccharide SEP from eggs of sea urchin Strongylocentrotus nudus, and to determine its purity, molecular weight and immunological activity in vitro. Methods The orthogonal design was employed to obtain the best possible combination of the critical parameters for polysaccharide extraction. By ultrafiltration, DEAE Sepharose Fast Flow anion-exchange chromatography and Sephacryl S-400 gel filtration chromatography, the deproteinated crude polysaccharide was purified. The homogeneity of SEP was proved by HPLC, polyacrylamide gel electrophoresis and paper chromatography. Its molecular weight was determined by HPGPC in reference to standard T-series Dextran. Lymphocyte proliferation assay was made to investigate the immuno-modulating activity of SEP. Results and Conclusion The results indicated SEP was a homogeneous polysaccharide. Its molecular weight was about 1950KD. SEP increased remarkably spleen lymphocyte proliferation. The homogeneous polysaccharide SEP showed significantly immunological activity in vitro.