大菱鲆（Scophthalmus maximus L.）中IgM样免疫球蛋白的纯化与特性分析
Purification and characterization of IgM-like immunoglobulin from turbot (Scophthalmus maximus L.).
作者信息
Kofod H, Pedersen K, Larsen J L, Buchmann K
机构信息
Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
出版信息
Acta Vet Scand. 1994;35(1):1-10. doi: 10.1186/BF03548350.
A total of 40 turbot (Scophthalmus maximus) were immunized 3 times during a 3 months period using DNP-HSA whereafter serum samples were collected and pooled. Specific immunoglobulins (Ig) were affinity purified on an agarose column with immobilized DNP-BSA and further purified by gel filtration whereafter monospecific rabbit anti Ig serum was generated. Size exclusion chromatography and non-reduced SDS-PAGE indicated a MW of 8-900 kDa of the dominant antigen binding proteins from turbot serum. Reduced SDS-PAGE showed this fraction to be composed of disulphide linked heavy and light chains with MWs of 79 and 27-29 kDa, respectively, indicating a tetrameric structure. Isoelectric focusing of the 800-900 kDa Ig showed several bands between pH 5.5 and pH 5.8. Mean Ig concentration in serum of 10 turbot was measured to 6.48 mg/ml (SD 5.4) using rocket immunoelectrophoresis. Low molecular weight antigen binding molecules were copurified with the dominating immunoglobulins with an estimated MW of 500 kDa. Reducing SDS-PAGE of this fraction revealed molecules with MWs of 97, 79, 57, 29, and 27 kDa.
在3个月的时间内，使用二硝基苯基人血清白蛋白（DNP-HSA）对总共40条大菱鲆（Scophthalmus maximus）进行3次免疫，之后收集血清样本并合并。特异性免疫球蛋白（Ig）在固定有DNP-牛血清白蛋白（DNP-BSA）的琼脂糖柱上进行亲和纯化，并通过凝胶过滤进一步纯化，之后制备单特异性兔抗Ig血清。尺寸排阻色谱和非还原十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）表明，大菱鲆血清中主要抗原结合蛋白的分子量为8 - 900 kDa。还原SDS-PAGE显示该组分由二硫键连接的重链和轻链组成，重链和轻链的分子量分别为79 kDa和27 - 29 kDa，表明其为四聚体结构。对800 - 900 kDa Ig进行等电聚焦，在pH 5.5至pH 5.8之间显示出几条条带。使用火箭免疫电泳法测得10条大菱鲆血清中的平均Ig浓度为6.48 mg/ml（标准差为5.4）。低分子量抗原结合分子与估计分子量为500 kDa的主要免疫球蛋白一起被共纯化。对该组分进行还原SDS-PAGE，显示出分子量为97、79、57、29和27 kDa的分子。
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