Heavy metals in three wastes are discharged into water by a variety of means with the industry development such as mine, smelting and electroplating, which lead to pollution of aquatic environments and threaten human health and biological survival seriously. Cadmium(Cd) is one of the main marine pollutants. As Cd can accumulate in different tissues of shellfish through food chains, which threaten the quality and safety of shellfish foods, it is extremely necessary to research and monitor conditions of shellfish contamination with Cd. Cadmium-metallothioneins (Cd-MTs) are important biomarker used for indicating cadmium pollution in aquatic environment. The research on these Cd-MTs not only is propitious to establish Cd-MT immunoassay method, but also provides the theoretical basis for technology development in monitoring heavy metal pollution. Cd-MT was induced to synthesize by injecting different concentrations of CdCl2(0 -0.8 mg/L)in Pteria penguin and was isolated and purified from whole tissues of P. penguin by tissue homogenization, freezing centrifugation, heat treatment and Sephadex G-50 gel column chromatography. The characteristic absorption of Cd-MT in crude extract by ultraviolet spectrophotometry was the strongest at 0.8 mg/L Cd2 +. The results showed that the content of Cd-MT in P. penguin increased with concentration of Cd2 +. The results from SDS-PAGE assay showed that molecular weight of purified Cd-MT and its dimer were 9 ku and 18 ku respectively. Then Cd-MT was coupled to bovine serum albumin (BSA) with glutaraldehyde and polyclonal antiserum by immunizing rabbits with Cd-MT-BSA was prepared. The titer of antiserum was measured by indirect ELISA assay and reached 1:12 800. IgG ( molecular weight is about 35 ku) in the antiserum was obtained by methods including saturated ammonium persulfate precipitation and DEAE-Sephadex A50 column chromatography. ELISA assay showed that the IgG from antiserum could react to Cd-MT of P. martensi as well as Cd-MT of P. penguin. The study will lay a foundation for establishing an indirect ELISA method of Cd-MT in shellfish and meanwhile, afford technological and theoretical support for exploitation of Cd-MT test kit used in shellfish.%镉-金属硫蛋白(Cd-MT)是水生环境中重金属镉污染的重要生物标志物,对该蛋白的研究不仅有利于Cd-MT免疫学检测方法的建立,也为水生环境重金属污染监测技术的开发提供理论依据.采用不同浓度CdCl2(0-0.8mg/L)胁迫诱导企鹅珍珠贝合成Cd-MT,通过组织捣碎、冷冻离心、热处理、SephadexG-50凝胶柱层析等方法从企鹅珍珠贝全脏器中纯化得到Cd-MT.紫外吸收显示,在CdCl2浓度为0.8mg/L时,MT粗提液中的Cd-MT特征吸收最大,表明贝体内的Cd-MT含量随着CdC12胁迫浓度的增大而增加.SDS-PAGE显示纯化后的Cd-MT及其二聚体分子量约为9ku和18ku.采用戊二醛法将获得的Cd-MT与牛血清白蛋白(BSA)偶联后免疫家兔,制备了兔抗企鹅珍珠贝Cd-MT多克隆抗血清.通过间接ELISA法检测其抗血清效价为1:12800.经饱和硫醚酸铵法和DEAE-SephadexA50亲和层析纯化得到分子量约为35ku的高纯度免疫球蛋白G(IgG).ELISA免疫鉴定显示,该IgG不仅能识别企鹅珍珠贝的Cd-MT,还与马氏珠母贝的Cd-MT有明显的免疫交叉反应.
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