摘要
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。
R-phycoerythrin（R-PF） was highly purified from red alga（ Bangia fusco-purpurea）by ammonium sulfate fractionation and DEAE- Sepharose anion- exchange column chromatography, the purity rate （A565/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 I gG 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.
出处
《食品工业科技》
CAS
CSCD
北大核心
2012年第17期147-150,共4页
Science and Technology of Food Industry
基金
福建省重大科研专项(2010NZ0001-3)
福建省自然科学基金(2010J01044)
关键词
R-藻红蛋白
荧光探针
交联
免疫检测
R-phycoerythrin
fluorescent probes
crosslinking
immunization detection
