用Protein A亲和层析法快速分离纯化鲫血清IgM方法的建立和应用

Rapid purification of serum IgM from crucian carp,Carassius auratus by Protein A agarose affinity chromatography

采用Protein A亲和层析法对鲫Carassius auratus血清中的IgM进行快速分离纯化,所得产物用聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blotting)进行分析检测。结果表明:采用Protein A亲和层析法可以很好地分离到高纯度的鲫血清IgM,电泳条带中重链和轻链清晰可辨,重链、轻链的相对分子质量分别为85 000、25 000左右,无明显杂带;利用纯化的鲫血清IgM免疫小鼠,获得了高效价抗IgM抗血清,可以特异性识别鲫血清和黏液中IgM重链。应用间接ELISA方法对浸泡免疫后的鲫血清和皮肤黏液中抗体的动态进行检测,结果显示:鲫皮肤黏液中的抗体滴度在免疫后第6天达到峰值,血清中抗体滴度在免疫后第15天达到峰值,前者高峰期出现较早,但持续时间短,后者高峰期出现较晚,但持续时间较长。本试验中所建立的Protien A亲和层析法为鱼类抗体制备、病原检测及免疫学相关研究提供了一种便捷的方法。

In present study,serum IgM from crucian carp,Carassius auratus,was purified rapidly using affinity chromatography with Protein A Agarose and then analyzed by SDS-PAGE.The results showed that high purity of serum IgM was separated using affinity chromatography technique,and about 85 000 of heavy chain and 25 000 of light chain was clearly detected in the IgM by SDS-PAGE without any other obvious bands.High titer of antisera which can detect heavy chain of IgM was obtained in kunming mice injected the purified IgM from both skin mucus and serum in the crucian carp.Furthermore,indirect ELISA method for evaluation of the dynamic change in the IgM of crucian carp after immersion vaccination revealed that the peak of the antibody titer detected from skin mucus achieved occurred 6 days after immunization in short duration,whereas the peak of the antibody titer detected from serum was observed 15 days after immunization with a longer duration.The establishment of affinity chromatography technique on IgM purification using Protein A agarose provides a convenient and rapid method for fish serum purification and further immunity related tests.