色谱

色谱

• 研究论文 • 上一篇    下一篇

琼脂糖凝胶CL-6B亲和色谱法一步纯化蚯蚓半乳凝素

陈义烘,黄慧敏,李任强

  

  1. Department of Biotechnology, Jinan University, Guangzhou 510632, China
  • 收稿日期:2006-11-13 修回日期:2006-12-18 出版日期:2007-05-30 发布日期:1985-09-25
  • 通讯作者: 李任强

Single-Step Purification of Galectin in Earthworm Using
Sepharose CL-6B Affinity Chromatography

CHEN Yihong, HUANG Huimin, LI Renqiang   

  1. Department of Biotechnology, Jinan University, Guangzhou 510632, China
  • Received:2006-11-13 Revised:2006-12-18 Online:2007-05-30 Published:1985-09-25

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摘要：

环节动物的S型凝集素在结构和生化性质上都有别于一般的S型凝集素，其在抗癌等研究方面的潜在价值重大。根据环节动物S型凝集素的性质,采用惰性分子筛填料Sepharose CL-6B（琼脂糖凝胶CL-6B）作为亲和色谱介质对蚯蚓S型凝集素进行了纯化。以2 mmol/L乙二胺四乙酸（EDTA）-MEPBS (4 mmol/L β-巯基乙醇,150 mmol/L NaCl,20 mmol/L 磷酸盐，pH 7.2)溶液作为平衡液，以氨水(150 mmol/L,pH 10.5)作为洗脱液得到的蛋白质经SDS-PAGE（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳）、凝血实验及荧光检测等证明了其即为蚯蚓S型凝集素。该方法只需一个步骤即可从蚯蚓提取液中分离到纯的S型凝集素,比传统方法更加快捷高效,优越性明显。该方法的应用将有利于对环节动物S型凝集素的深入研究。

关键词: S型凝集素, 亲和色谱, 琼脂糖凝胶CL-6B, 蚯蚓 , 一步纯化

Abstract:

The S-type lectins in annelida are different in molecular structure and biochemical properties from that of common galectin and are valuable in anticancer study. Based on their specificity, Sepharose CL-6B affinity chromatography was adopted in this study to separate the S-type lectin in annelida from earthworms, in which EDTA-MEPBS (2 mmol/L EDTA, 4 mmol/L β-mercaptoethanol, 150 mmol/L NaCl, 20 mmol/L phosphate, pH 7.2) was used as an equilibrium solution and aqueous ammonia solution (150 mmol/L, pH 10.5) as an eluent. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the relative molecular mass of the obtained protein in this purification procedure was about 32000 and it could be further separated into fractions with relative molecular mass about 15000. Hemagglutination experiment and fluorescence analysis demonstrated that this protein possessed the characteristics of agglutinin and its complex with lactose. This indicated that the target protein was one of the S-type lectins in annelida. This study may offer a novel and rapid method for purifying the S-type lectins in annelida in large scale.

Key words: affinity chromatography, earthworm , galectin, single-step purification, Sepharose CL-6B