The crude extract of phycobiliproteins was prepared from thallus of Porphyra yezoensis with combination of dissolving-bulging and tissue-homogenator methods.Effects of solvent volume,treating time,ammonium sulphate precipitation time,and hydroxylapatite(HA)chromatography on purity and yield of phycobiliprotein were studied.The results showed that the optimum conditions for purification,including the ratio of thallus to buffer(1:5),treatment with dissolving-bulging,precipitation with ammonium sulphate for four times,all of which increased the purity of phycoerythrin and phycocyanin up to 1.71 and 0.98,respectively.Due to HAP chromatography according to Siegelman,the phycoerythrin and phycocyanin purity increased to 4.73 and 4.42,with the yield of 0.144% and 0.042%.

To optimize the chemical conjugation between R-phycoerythrin (R-PE) and antibody, different molar ratios of the heterobifunctional reagent N-succinimidyl-3-2-pyridyldithio propionate (SPDP) to R-PE were tested for R-PE derivation into R-PE-PDP, and different molar ratios of dithiothreitol (DTT) to IgG were tested for IgG thiolation. The results showed that in terms of best product yields determined by ultraviolet (UV) spectrophotometry, the optimal molar ratio of SPDP to R-PE was 40:1 for PE derivation, and that of DTT to IgG was 500:1 for thiolation. R-PE-labeled secondary antibody was produced by cross-linking PE-PDP and thiolated IgG. After further purification, UV spectra and native polyacrylamide gel electrophoresis determined its high purity and molecular weight. Finally, in conjunction with antigen-specific first antibodies, the R-PE-labeled IgG was applied in fluorescence immunoassays as secondary antibody and successfully detected antigens spotted on nitrocellulose membrane as well as intracellular antigen in SMCC-7721 cells. This study provides a feasible method of fluorescence antibody preparation from R-PE of Porphyra yezoensis and demonstrates high fluorescent labeling efficiency and good immunologic reactivity of the product.

Objective:This paper presents pilot study on extraction and purification of costliness phycobiliproteins abound in Spirulina maxima.Methods:tissue-trituration method,freezing/unfreezing method and ultrasonic method were applied and compared to prepare crude extracts.Further purification was performed by means of ammonium sulphate precipitation and hydroxylapatite(HA)column chromatogram.Purified protein were examined through absorption spectrum and SDS-PAGE.Results:The results showed that ultrasonic method was the optimal method for extracting phycobiliproteins, and twice hydroxylapatite(HA)column chromatograms have to be performed to obtain fine product.Conclusion:considering cost of economy,manpower and time,this technology is useful in large-scale isolation and purification of phycobiliproteins in Spirulina maxima.

We optimized the chemical conjugation between R-phycoerythrin and antibody.First,the R-PE(R-phycoerythrin)was derived with beterobifunctional reagent SPDP(N-succinimidyl-3-2-pyridyldithio propionate)and antibody was thiolated with DTr(dithiothreitol).Second,we determined the effects of the different molar ratio of SPDP to R-PE for the derivation and DTT to IgG for the thiolation.The results showed that the optimum molar ratio of SPDP to R-PE for derivation was 40:1,and DTT to IgG for thiolation was 500:1.The conjugation between R-phycoerythin and antibody was further done for fluorescence probe preparation. R-phycoerythin was conjugated with IgG and formed a probe complex by whole wavelength scanning,electrophoresis(Native-PAGE) and fluorescence microscope observation.