人血浆中MBL-MASP复合物的纯化与分离

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (12): 1373-1377.

人血浆中MBL-MASP复合物的纯化与分离

陈月, 张丽芸, 陈政良

南方医科大学免疫学教研室, 广东, 广州, 510515

出版日期:2004-12-20 发布日期:2004-12-20
基金资助:
收稿日期:2004-7-14。
基金项目:国家自然科学基金(30371310);广东省自然科学基金研究团队项目(015003);
作者简介:陈月(1964- ),女,黑龙江齐齐哈尔人,在读博士研究生,副主任检验师,主要从事天然免疫研究,电话:020-61640114-89280,E-mail:chenyue@fimmu.com.
通讯作者:陈政良,电话:020-61640114-89280,E-mail:zhlchen@fimmu.com

Purification and separation of mannan-binding lectin (MBL) and MBL-associated serine proteases complex from human plasma

CHEN Yue, ZHANG Li-yun, CHEN Zheng-liang

南方医科大学免疫学教研室, 广东, 广州, 510515

Online:2004-12-20 Published:2004-12-20

摘要/Abstract

摘要： 目的从人血浆中分离纯化甘露聚糖结合凝集素(MBL)和MBL相关丝氨酸蛋白酶(MASP)。方法用非活化Sepharose4B两步亲和层析纯化MBL-MASP复合物,再以SephacrylS-300柱凝胶过滤将MBL和MASP分离。在纯化MBL-MASP复合物的整个过程中,除凝胶过滤缓冲液外,均加入丝氨酸蛋白酶抑制剂苯甲磺酰氟(PMSF)和金属蛋白酶抑制剂1,10-邻二氮杂菲(1,10-phenanthroline),并控制温度在4 ℃。结果获得高度纯化的MBL和酶原形式的MASP。SDS-PAGE和Westernblotting表明所纯化的MBL相对分子质量为28000和32000肽链构成的功能性多聚体;配体结合测定和酵母菌凝集试验证实所纯化的MBL具有生物学活性。结论建立了一种比较简便的同时分离纯化MBL和MASP酶原的方法。

Abstract: Objective To separate mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) from human plasma. Methods A two-step affinity chromatography on underivatized sepharose 4B was employed for purification of MBL-MASP complex, followed by gel filtration on a Sephacryl S-300 column for separation of MBL and MASPs from the complex. The purification procedures were performed at 4℃ with the addition of two proteolytic inhibitors, phenyl methylsulfonyl fluoride and 1,10-phenanthroline during affinity chromatography but not in the gel filtration buffer. Results Preparations of highly purified MBL and proenzyme MASPs were obtained. The purified MBL was shown by SDS-PAGE and Western blotting to be a functional multimer composed of 28 000 and 32 000 peptide chains, with high bioactivity as demonstrated by ligand-binding assay and yeast agglutination experiment. Conclusion A simple and convenient procedure is established successfully for the purification of MBL and the proenzyme MASPs.

中图分类号:

R392-33

陈月, 张丽芸, 陈政良. 人血浆中MBL-MASP复合物的纯化与分离[J]. 南方医科大学学报, 2004, 24(12): 1373-1377.

CHEN Yue, ZHANG Li-yun, CHEN Zheng-liang. Purification and separation of mannan-binding lectin (MBL) and MBL-associated serine proteases complex from human plasma[J]. Journal of Southern Medical University, 2004, 24(12): 1373-1377.

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