R-藻蓝蛋白的分离纯化及抗食物过敏活性研究

Research on the Purification and Anti-food Allergic Activity of R-phycocyanin

为探究R-藻蓝蛋白抗过敏功效,以坛紫菜为原料,通过25%～35%硫酸铵盐析和DEAE-Sepharose Fast Flow、Sephacryl S-200 HR两步柱层析获得了高纯度的目的蛋白(A620/A280>6.0).SDS-PAGE显示,该蛋白质由分子质量为17.7 ku和20.5 ku的两个亚基组成,紫外可见分光光度法测得其在620 nm、555 nm有两个特征吸收峰,可确认其为R-藻蓝蛋白.热稳定性和pH值稳定性实验结果显示,R-藻蓝蛋白在低于35℃、pH=4.0～9.0条件下光谱性质相对稳定.动物实验结果表明,100μg/mL高纯度R-藻蓝蛋白能够显著降低小鼠血清中IgE水平;细胞实验结果表明,40μg/mL R-藻蓝蛋白能够降低细胞因子IL-4、IL-13的蛋白质分泌量;基因表达量分析结果显示,R-藻蓝蛋白能够降低IL-4在淋巴细胞中的表达量.可见,坛紫菜R-藻蓝蛋白通过促使CD4+T cell向TH1型转化发挥抗食物过敏活性.

In order to explore the anti-food allergic effect of R-phycocyanin, high purity （A620/A280 〉 6. 0） of interest protein from Porphyra haitanensis was acquired after 25 % - 35 % ammonium sulfate fraction-ation, DEAE-Sepharose Fast Flow and Sephacryl S-200 HR column chromatographies. SDS-PAGE showed that the protein consisted of two different subunits, with molecular weight of 17. 7 ku and 20. 5 ku, respec-tively. Two characteristic absorption peaks at 620 nm and 555 nm as detected by ultraviolet spectrophotometer were used to identify R-phycocyanin. Thermal and pH stability test demonstrated that the spectroscopic prop-erties of R-phycocyanin was stable under the conditions of 〈 35 ℃, pH = 4. 0 - 9. 0. Animal experiment showed that R-phycocyanin （100 μg/mL） could reduce the igE level in mouse serum significantly. In vitro cellular experiment, revealed that R-phycocyanin （40 μg/mL） could reduce the protein secretion of IL-4 and IL-13 in protein level. Gene expression analysis using lymphocytes showed that R-phycocyanin could re-duce the expression of IL-4. In conclusion, R-phycocyanin could reduce food allergic reaction via skewing the immunological function of CD4^＋ T cell toward TH1 activity.