用鼠红细胞膜纯化中华绒螯蟹血清凝集素的方法  

Purification of Eriocheir sinensis serum agglutinin using mouse erythrocyte membrane

作　　者：刘雪兰[1] 余为一[1] 李槿年[1] 

机构地区：[1]安徽农业大学畜牧水产学院,安徽合肥230036

出　　处：《中国水产科学》2004年第6期519-524,共6页Journal of Fishery Sciences of China

基　　金：安徽省自然科学基金项目(99041355).

摘　　要：本研究建立鼠红细胞膜吸附法纯化中华绒螯蟹(Eriocheir sinensis)血清凝集素的方法,并与硫酸铵盐析、凝胶过滤层析和离子交换层析等其他提取方法进行比较。实验结果表明,中华绒螯蟹血清经鼠红细胞膜吸附后,再由EDTA把结合的凝集素从膜上解离,可以获得纯化的高活性凝集素。该纯化物在PAGE中出现2个清晰的区带,一个是大分子的b带,另一个则是由3种大小相近的小分子组成的c区带;在SDS-PAGE中,则显示出3个分子量接近、约为100 000的条带。经50％饱和硫酸铵盐析或用Sephadex G200凝胶过滤层析法可浓缩但不能纯化中华绒螯蟹血清凝集素。经DEAE-Sephadex A50离子交换层析法可获得2个蛋白峰,其中只有1个峰有凝集活性,在PAGE中表现为a蛋白带。凝集活性较高、分子量较大的蛋白带。上述实验结果表明,应用鼠红细胞膜法可纯化中华绒螯蟹血清凝集素。Eriocheir sinensis, as a kind of crustacean animals, is one of the important aquatic products in Chi-na.Since lack of specific immune system,crustacean animals require unspecific immune factors,such as ag-glutinin,to resist pathogenic microbes. Agglutinin is a big family composing of different molecules.So far, the serum agglutinin in these animals has not been well identified. The aim of this study was to purify Eriocheir sinensis serum agglutinins and research their characters.Firstly,a method was established to purify agglutinin molecules of Eriocheir sinensis serum, which was named mouse erythrocyte membrane adsorption ( MEMA) . Briefly, after a reaction of Eriocheir sinensis serum (0.5 mL) with prepared mouse erythrocyte membrane (0. 1 g of wet weight) for 2 h and following a centrifugation,the precipitated membrane was suspended with 0.5 mL EDTA (4 mol/L,pH5.0) and incu-bated for 1 - 12 h. Then a centrifugation was carried out and the supernatant was dialyzed against PBS to remove EDTA. The results of agglutination test showed that the samples kept the same activity treated with EDTA for 1 h as well as 12 h. These purified molecules had two sections in a polyacrylamide gel elec-trophoresis (PAGE) map,one of them contained three bands,named band c, with the similar lower molecule weight (MW) and other was a single band, named band b,with higher MW. However,in SDS - PAGE, they had one section containing three bands with the similar MW about 100000.Secondly, we used other methods such as saturated ammonium sulfate precipitation, gel chromato-graphy with Sephadex G200 and ion-exchange chromatography with DEAE Sephadex A50 to gain the agglutinins. The ammonium sulfate solution with final saturation of 33% ,50% and 75% was used to precipi-tate the Eriocheir sinensis serum, successively. After centrifugation, the precipitates were dialyzed against PBS (pH 7.2,0.01 mol/L) for 48 h to remove ammonium sulfate. The highest activity of agglutinin was found in the sample used by 50% saturated ammonium sulfate, in which

关 键 词：中华绒螯蟹 凝集素 鼠红细胞膜吸附 纯化 

分 类 号：Q959.223[生物学—动物学]