水产学报2011，Vol.35Issue(6)：816-823,8.DOI:10.3724/SP.J.1231.2011.17413
企鹅珍珠贝镉金属硫蛋白的分离纯化及其多克隆抗体的制备
Purification and polyclonal antibody preparation of Cd-MT from Pteria penguin
摘要
Abstract
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.关键词
企鹅珍珠贝/镉金属硫蛋白/多克隆抗体Key words
Pteria penguin/ Cd-MT/ polyclonal antibodies分类
农业科技引用本文复制引用
吴晓萍,廖爱琳,章超桦,廖艳,卢虹玉,杨捷..企鹅珍珠贝镉金属硫蛋白的分离纯化及其多克隆抗体的制备[J].水产学报,2011,35(6):816-823,8.基金项目
现代农业产业技术体系(CARS-48-07B):广东省科厅农业攻关项目(2010B 020313005):广东海洋大学自然科学基金(2010) （CARS-48-07B）
