Preparation of R-phycoerythrin from red alga and its application for antibody labeling and detection[J]. Science and Technology of Food Industry, 2012, (17): 147-150. DOI: 10.13386/j.issn1002-0306.2012.17.001
Citation: Preparation of R-phycoerythrin from red alga and its application for antibody labeling and detection[J]. Science and Technology of Food Industry, 2012, (17): 147-150. DOI: 10.13386/j.issn1002-0306.2012.17.001

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

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  • Received Date: January 15, 2012
  • 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.
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  • [1]
    陈国珍, 黄贤智, 郑朱梓, 等.荧光分析法[M].北京:科学出版社, 1990:1-28.
    [2]
    Jason J, Larned J.Single-cell cytokine profiles in normal humans:comparison of flow cytometric reagents and stimulation protocols[J].J Immunol Methods, 1997, 207:13-22.
    [3]
    Winterrowd G E, Chin J E.Flow cytometric detection of antigen-specific cytokine responses in lung T cells in a murine model of pulmonary inflammation[J].J Immunol Methods, 1999, 226:105-118.
    [4]
    Glazer A N.Phycobilisome, a macromolecular complex optimized for light energy transfer[J].Biochim Biophys Acta, 1984, 768:29-51.
    [5]
    Liu L N, Chen X L, Zhang X Y, et al.One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata[J].J Biotechnol, 2005, 116:91-100.
    [6]
    Gantt E, Lipschultz C A, Grabowski J, et al.Phycobilisomes from blue green and red algae:isolation criteria and dissociation characteristics[J].Plant Physiol, 1979, 63:615-620.
    [7]
    Sun L, Wang S M, Gong X Q, et al.Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica[J].Protein Expres Purif, 2009, 64:146-154.
    [8]
    Soni B, Kalavadia B, Trivedi U, et al.Extraction, purification and characterization of phycocyanin from Oscillatoria quadripunctulata-isolated from the rocky shores of Bet-Dwarka, Gujarat, India[J].Process Biochem, 2006, 41:2017-2023.
    [9]
    Kaixian Q, Franklin M, Borowitzka M A.The study for isolation and purification of R-phycoerythrin from a red alga[J].Biotechnol Appl Biochem, 1993, 43 (2) :133-140.
    [10]
    陈小强.坛紫菜中R-藻红蛋白的分离纯化、理化性质及生物活性的研究[D].杭州:浙江大学, 2004.
    [11]
    Rossano R, Ungaro N, D’Ambrosio A, et al.Extracting and purifying R-phycoerythrin from Mediterranean red algae Corallina elongate Ellis&Solander[J].J Biotechnol, 2003, 101:289-293.
    [12]
    顾铭, 吴萍, 戚艺华, 等.藻胆蛋白荧光探针及其标记[J].生命科学研究, 2001, 5 (2) :109-113.
    [13]
    Zhou M, Cai C, Liu J X, et al.Optimization of IgG conjugation with R-phycoerythrin from Porphyra yezoensis[J].J Appl Phycol, 2010, 22:459-464.
    [14]
    Laemmli U.Cleavage of structural proteins during the assembly of the head of the bacteriophage T4[J].Nature, 1970, 277:680-685.
    [15]
    洪秀庄, 孙曼霁.蛋白质连接技术[M].北京:中国医药科技出版社, 1992, 34-35.
    [16]
    Towbin H, Staehelin T, Gordon J.Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications[J].Proc Natl Acad Sci USA, 1979, 76:4350-4354.
    [17]
    Glazer A N, Stryer L.Fluorescent phyeobili protein conjugates for analyses of cells and molecules[J].J Cell Biol, 1982, 93:981-986.
    [18]
    颜世敢, 朱丽萍, 张玉忠, 等.藻红蛋白标记抗鸡IgG荧光抗体的高效制备[J].中国预防兽医学报, 2009, 31 (2) :127-131.
    [19]
    Schubert-Ullrich P, Rudolf J, Ansari P, et al.Commercialized rapid immunoanalytical tests for determination of allergenic food proteins:an overview[J].Anal Bioanal Chem, 2009, 395 (1) :69-81.
    [20]
    Faste C K, P1assen C.Quantitative sandwich ELISA for the determination offish in foods[J].J Immunol Methods, 2008, 329 (1-2) :45-55.
    [21]
    Sun M, Liang C, Gao H, et al.Detection of parvalbumin, a common fish allergen gene in food, by real-time polymerase chain reaction[J].J AOAC Int, 2009, 92 (1) :234-240.

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