罗非鱼和斑点叉尾鮰血清IgM的纯化及兔抗血清的制备

Purification of serum immunoglobulin(IgM) from tilapia(Oreochromis niloticus) and channel catfish(Ictalurus punctatus) and preparation of their anti-IgM sera in rabbit

为深入了解罗非鱼和斑点叉尾鮰免疫机理及建立相关免疫检测方法,采用rProtein A Sepharose亲和层析一步法纯化罗非鱼和斑点叉尾鮰血清免疫球蛋白(IgM),并制备其兔抗血清。结果表明,rProtein A Sepharose亲和层析法可以较好地分离获得高纯度的罗非鱼和斑点叉尾鮰血清IgM,通过SDS-PAGE电泳检测,发现罗非鱼血清IgM重链和轻链分子量分别为88.0、21.0 kDa,斑点叉尾鮰血清IgM重链分子量为101.0 kDa。以纯化的罗非鱼和斑点叉尾鮰血清IgM为抗原,制备其兔抗血清,间接ELISA检测其效价分别为1∶32000和1∶16000;Western blotting检测分析,发现罗非鱼和斑点叉尾鮰的兔抗血清分别在88.0和101.0 kDa附近各出现1条反应条带,说明其兔抗血清具有免疫活性。

The serum immunoglobulins （IgM） of tilapia （Oreochromis niloticus） and channel catfish （Ictalurus punctatus） were purified using rProtein A Scpharose affinity chromatography, a one-step purification method. The purified IgM was applied to rabbit for preparation of anti-IgM sera. The results showed that the IgMs from two kinds of fish were well separated from each serum and were obtained at high purity by the rProtein A Sepharose affinity chromatography. The molecular weights of tilapia＇s heavy chain and light chain was 88.0 and 21.0 kD, respectively. While the molecular weight of channel catfish＇s heavy chain was 101.0 kD. Sera anti-IgM of Oreochromis niloticus and Ictalurus punctatus were prepared by repeatedly immunizing the New Zealand rabbits with purified IgM, and the antibody activity of anti-sera was tested by Western Blotting. The titers of anti-sera reached 1：32000 and 1：16000, respectively, when detected by indirect ELISA method. The Western Blotting results showed that there was a band nearby 88.0 and 101.0 kD for rabbit anti-sera of tilapia and channel catfish, respectively, which proved that the obtained anti-sera had immune activity.