红毛藻R-藻红蛋白的高效制备以及抗体标记与检测

采用硫酸铵盐析和DEAE-Sepharose阴离子交换柱层析相结合,从红毛藻（Bangia fusco-purpurea）中分离纯化到纯度系数（A565/A280）大于6.0的R-藻红蛋白。SDS-PAGE结果显示,其亚基分子量分别为19、21ku。用适宜摩尔浓度的异型双功能交联剂N-琥珀酰亚氨基-3-2-吡啶基二硫丙酸醇（SPDP）将纯化的R-藻红蛋白与羊抗小鼠IgG抗体交联,利用SDS-PAGE和荧光检测鉴定R-藻红蛋白与抗体的交联效果。结果显示:R-藻红蛋白能与羊抗小鼠IgG成功交联。分别采用Dotblot和Westernblot对R-藻红蛋白标记的抗体作为二次抗体,小鼠抗鲢鱼主要过敏原小清蛋白单克隆抗体为一次抗体;对鱼类小清蛋白进行免疫检测,结果显示:R-藻红蛋白标记的抗体能检测相关抗原的存在,且有良好的特异性。利用藻红蛋白荧光探针可缩短免疫杂交的检测时间,简化操作过程。 

Preparation of R-phycoerythrin from red alga and its application for antibody labeling and detection

R-phycoerythrin(R-PE)was highly purified from red alga(Bangia fusco-purpurea)by ammonium sulfate fractionation and DEAE-Sepharose anion-exchange column chromatography,the purity rate(A 565 /A280)of R-PE was over 6.0.SDS-PAGE demonstrated that relative molecular masses of R-PE subunits were about 19 and 21ku,respectively.Optimum concentration of heterobifunctional reagent N-succinimidyl-3-2-pyridyldithio propionate(SPDP)was used to polymerize goat anti-mouse IgG antibody and the cross-linking was identified using SDS-PAGE and fluorescence analysis.R-PE was successfully crosslinked with goat anti-mouse IgG as secondary antibody.Detection of major allergen parvalbumin in fish using R-PE labelled IgG were carried out by Dot blot and Western blot together with mouse anti-silver carp parvalbumin as primary antibody.The results demonstrated that R-PE labelled IgG could effectively detect the existence of parvalbumin and revealed good immunological specificity.The application of R-phycoerythrin as fluorescent probe is expected to shorten the detection time of immune hybridization,and simplify the operation processes.