锯缘青蟹凝集素的提取及单克隆抗体制备  

Isolation and preparations of monoclonal antibodies against lectin from mud crab,Scylla serrata

作　　者：郝珂[1,2] 钱冬[2] 刘问[2] 潘清清[2,3] 

机构地区：[1]宁波大学生命科学与生物工程学院,浙江宁波315211 [2]浙江省淡水水产研究所,浙江湖州313001 [3]浙江省三门县海洋与渔业局,浙江三门317100

出　　处：《水产学报》2009年第4期590-596,共7页Journal of Fisheries of China

基　　金：浙江省重大科技攻关项目(2005C12038);浙江省自然科学基金(Y307479)

摘　　要：凝集素是无脊椎动物非特异性免疫系统的重要组成成份，可凝集外来病原菌、进行非己识别、调理和介导血细胞吞噬。开展了锯缘青蟹血清凝集素的提取、单克隆抗体制备及凝集素定位研究，以N-乙酰基葡萄糖胺（N—Acetyl—D—glucosamine，GlcNac）为配基制备了Sepharose4B亲和层析柱，可选择性结合青蟹血清凝集素；青蟹血清通过亲和层析柱后，以0．45mol／LNaCl洗脱，得到有较高凝集活性的单一洗脱峰，该洗脱峰凝集素比活为血清凝集活性的32．6倍，SDS—PAGE显示存在87ku和79ku主要蛋白成分；以纯化青蟹凝集素免疫BALB／c小鼠，经细胞融合、单抗筛选获得BFS-2等7株可稳定分泌抗凝集素单抗的小鼠杂交瘤细胞株，单抗腹水的ELISA效价为1：10^4～1：10^5；单抗可特异性抑制青蟹凝集素的血凝，血凝抑制效价为1：32～1：64，亚型鉴定表明7株单抗均为kG1型，免疫印迹表明单抗可特异性结合87ku、79ku蛋白；采用凝集素单抗间接免疫荧光法进行了青蟹血淋巴细胞染色，结果显示凝集素主要分布于颗粒细胞的细胞质颗粒和透明细胞的细胞膜上。Lectin plays an important role in non-specific immunity of invertebrates by including bacterial agglutination, nonself-recognition, as opsonin and enhancing cell phagocytosis. In this paper, the author reported the purification and monoclonal antibodies （mAbs） preparation of lectin from mud crab, Scylla serrata, as well as the application of mAbs for lectin location by indirect immunofluorescence. Lectin was isolated from hemolymph of mud crab by affinity chromatography using Sepharose 4B coupled with N- Acetyl-D-glucosamine （GlcNac） which could specifically bind to mud crab lectin. The serum of S. serrata was applied to the column and the lectin binding to the column could be eluted by 0.45 mol/L NaCl Tris- HCl buffer with a single peak. The agglutinating specific activity of elution peak was equated 32.6 times of original serum, and SDS-PAGE showed the two main components of lectin with 87 ku and 79 ku. Seven hybridoma cell lines named BFS-2 etc., secreting antibodies against mud crab lectin, were prepared by hybridoma technique. Ascitic fluids of mAbs were 1 ： 10^4 - 1： 10^5 titers of indirect-ELISA by coated purified lectin. The mAbs could inhibit the hemagglutination of mud crab lectin with 1 ： 32 - 1 ： 64 titers. The Ig isotype for all seven mAbs were IgG1. The Western blots displayed that the mAbs specifically binded to the bands of 87 ku and 79 ku. Haemocytes of S. serrata were used for location of lectin by using indirect immunofluorescence technology; the results suggested that lectin is mainly distributed in the granules of granule hemocyte and the membrane of hyaline hemocyte.

关 键 词：锯缘青蟹 凝集素 亲和层析 单克隆抗体 间接免疫荧光 

分 类 号：S917[农业科学—水产科学]