大黄鱼血清IgM纯化及其兔抗血清的制备

Purification of serum IgM from large yellow croaker(Pseudosciaena crocea) and preparation of rabbit sera anti-IgM

分别用饱和硫酸铵二次盐析法和蛋白A亲和层析法对健康大黄鱼(Pseudosciaena crocea)血清中的免疫球蛋白(IgM)进行分离纯化,所得产物用SDS-PAGE进行检测。结果表明,蛋白A亲和层析法可以较好地分离到高纯度的大黄鱼血清IgM,产物的电泳胶中只有重链和轻链2个条带;饱和硫酸铵二次盐析法除了有这2个条带,还有很多杂带,而且蛋白A亲和层析法更为简便、快速,因此用蛋白A亲和层析法分离纯化IgM优于饱和硫酸铵二次盐析法;大黄鱼免疫球蛋白重链的分子量在76 kD左右;轻链分子量在28 kD左右。用纯化的大黄鱼IgM免疫实验兔,获得效价高达1∶40 960的兔抗鱼IgM血清。本实验所建立的蛋白A亲和层析法提取大黄鱼血清IgM可以方便、快捷地获得高纯度的产物,适合在实验室中纯化鱼类IgM。本研究所制备的兔抗大黄鱼IgM血清可为今后的相关研究工作打下基础。

The serum IgM of healthy large yellow croaker （Pseudosciaena crocea ） was purified by twice salt out of saturated （NH4）2SO4 and protein A-sepharose affinity chromatography. The products were examined by SDS-PAGE electrophoresis. The results showed that the product of protein A-sepharose affinity chromatography had only two bands （heavy chain and light chain） in SDS-PAGE, while the product of twice salt out of saturated （NH4）2SO4 had several other bands; moreover, using protein A-sepharose affinity chromatography to purify IgM was more simple, convenient and fast, so protein A- sepharose affinity chromatography was better than twice salt out of saturated （NH4）2SO4 in terms of the purification of IgM; the molecular weight of heavy chain and light chain of Pseudosciaena crocea IgM were 76 kD and 28 kD respectively. Sera anti-IgM of Pseudosciaena crocea had been prepared by repeatedly immunized New Zealand rabbits with purified igM, and the titers of anti-sera obtained up to 1 ： 40 960 examined by indirect ELISA. The results indicated that protein A-sepharose affinity chromatography was feasible in purification of IgM of Pseudosciaena crocea. The anti-sera obtained could be used in correlative studies in future.