【摘要】 运用Sephacryl S-200凝胶层析和HiTrap rProtein ASepharose亲和层析2种方法对牙鲆(Paralichthys olivaceus)血清免疫球蛋白进行分离纯化,结果表明,牙鲆免疫球蛋白分布于33%~50%的硫酸铵饱和溶液中,其中45%的分离效果最好。凝胶层析和亲和层析样品均出现2个蛋白峰,用还原SDS-PAGE检测确定牙鲆免疫球蛋白存在于第2个蛋白峰中。牙鲆免疫球蛋白重链分子量约为75.4 kD,轻链分子量约为29.9 kD和28.2 kD,推测牙鲆血清免疫球蛋白的分子量为836 kD。制备了兔抗牙鲆免疫球蛋白多克隆抗体,免疫双扩散法检测多克隆抗体效价为1∶32,免疫斑点法检测多克隆抗体效价至少为1∶1 600。运用免疫印迹法(Western-bloting)检测了兔抗牙鲆免疫球蛋白多克隆抗体的特异性,实验证明该抗体与牙鲆全血清中免疫球蛋白重链、轻链反应均成阳性。更多还原

【Abstract】 Japanese flounder,Paralichthys olivaceus,a species with high commercial value in north costal China,has been attacked by many diseases nowadays.Since it has an importance value both in theory and in practice usage,the immune system has been widely studied.In the last decade,it was well established that the teleost predominant immunoglobulins(Ig) have been shown to belong to a single class(IgM),and the studies on the teleost immune system have been benefited greatly from the production of poloclonal or monoclonal antibodies that have been used as specific markers.In this work,the purified Ig were isolated from serum of healthy flounder,and then used to produce polyclonal rabbit anti-flounder Ig antibody.The serum immunoglobulins of flounder were purified by means of Sephacryl-200 gel column chromatography and HiTrap rProtein A Sepharose affinity chromatography.The results showed that the immunoglobulins were precipitated in ammonium sulfate with saturation from 33% to 50%,with best effect in the 45% ammonium sulfate saturation.Both chromatography methods showed that there were two protein peaks and the immunoglobulins existed at the second peak that was detected by the dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) under reducing condition.Further analysis of the immunoglobulins showed that the molecular weight of flounder Ig was approximately 836kD,composing of one heavy chain weighing about 75.4kD,and two light chains weighing about 29.9kD and 28.2kD,respectively.A polyclonal rabbit against flounder antibody Ig was produced.The antiserum titer was evaluated in double immunodiffusion as 1:32 and in immunodotting assay as 1∶1600.Western blotting illuminated that the antibody had immunological activity by reaction with the heavy chain and light chains of flounder serum Ig and no cross-reaction with that of human.Western blotting proved the antiserum immunological specific activity and could be used in the further study on the immunolocatlisation of Ig-positive cells in lymphoid tissue of the flounder更多还原