Research For Basis And Application Of Phycoerythrin From Red Algea Bangia Fusco-purpurea

R-phycoerythrin was isolated and purified from Bangia fusco-purpurea after 35-50% ammonium sulfate fractionation followed by ion-exchange column chromatography on DEAE-Sepharose and the purity(A565/A280) of the phycoerythrin reached over 6.0. Absorption spectrum characteristic of the purified phycoerythrin showed that it is R-phycoerythrin, and its fluorescence emission maximum is 572 nm. The purified R-phycoerythrin was also identified with electrophoresis. SDS-PAGE together with fluorescent analysis demonstrated that relative molecular masses of subunits consisting of R-phycoerythrin were about 19,000, 21,000 Da, respectively. Only one single protein band appeared on Native-PAGE with coomassie brilliant blue staining, showing great purified effection.Transglutaminase was used to polymerize R-phycoerythrin to avidin. SDS-PAGE together with fluorescent analysis demonstrated that R-phycoerythrin was successfully crossinglinked avidin, and when the mole ratio of R-phycoerythrin to avidin was 1:20, the crosslinking efficiency reach the best. The fluorescent R-phycoerythrin conjugates applied in immunological detection was achieved by Dot blot. The results indicated that the fluorescent marker could detect the existence of biotin and antibody labelled biotin, showing better immunologic specificity and good fluorescent intensity.Cross-linking reaction of R-phycoerythrin and rabbit anti-soybean trypsin inhibitor(STI) IgG or goat anti-mouse IgG was performed by heterobifunctional reagent N-succinimidyl-3-2-pyridyldithio propionate(SPDP), respectively. Cross-linking consequences of R-phycoerythrin and IgG were evaluated using SDS-PAGE and fluorescence analysis. It turned out that R-phycoerythrin was successfully crosslinked rabbit anti-STI IgG or goat anti-mouse IgG, respectively. The fluorescent conjugates applied in immunological detection was achieved by Western blot or Dot blot. The results indicated that these fluorescent markers could detect the existence of the soluble antigen, showing better immunologic specificity. The better protocol of cross-linking of R-phycoerythrin and rabbit anti-STI as follows: the optimal molar ratio of SPDP to R-PE was 80:1; the optimal molar ratio of SPDP to rabbit anti-STI was 100:1.Optimum concentration of heterobifunctional reagent SPDP was used to polymerize anti-HBs/F, goat anti-mouse IgG antibody and mouse anti-rabbit antibody. The cross-linking was identified using SDS-PAGE and fluorescence analysis. R-phycoerythrin was successfully crosslinked with anti-HBs/F as primary antibody, goat anti-mouse IgG and mouse anti-rabbit IgG as secondary antibody. Detection of hepatitis B surface antigen using R-phycoerythrin labelled Anti-HBs/F was carried out by Dot blot. The results demonstrated that R-phycoerythrin labelled IgG could effectively detect 1.9 μg/mL of hepatitis B surface antigen and revealed good immunological specificity. 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 showed that R-PE labelled IgG could effectively detect 0.03 mg/mL of parvalbumin and revealed good immunological specificity. Detection of tropomyosin in Octopus using R-phycoerythrin labelled goat anti-mouse were carried out by Dot blot and Western blot together with rabbit anti-octopus tropomyosin as primary antibody. The results indicated that R-phycoerythrin labelled IgG could effectively detect 5.625 μg/mL of tropomyosin 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.