坛紫菜别藻蓝蛋白的纯化及多克隆抗体的制备

Purification of allophycocyanin from Porphyra haitanensis and prepraration of its polyclonal antibody

通过液氮研磨、超声波破碎、硫酸铵分级沉淀和DEAE-Sephadex-A-50柱层析等手段，建立了从坛紫菜叶状体体细胞中快速高效纯化别藻蓝蛋白的技术，并以该蛋白为抗原，通过动物免疫，制备了抗别藻蓝蛋白的多克隆抗体。电泳及Western印迹杂交分析的结果表明，纯化的别藻蓝蛋白已达到电泳纯，垴／A280达到3．5，计算蛋白回收率为70．5％。制备的多克隆抗体特异性强，效价高达1：100000。本研究的结果为进一步研究别藻蓝蛋白的功能和进行开发提供了基础材料和有用的工具。

In this study, a series of methods including grinding in the presence of liquid N2, ultrasonic disruption, ammonium sulfate precipitation and gel filtration chromatography on DEAE-Sephadex-A-50 were applied to isolation of allophycocyanin, a crucial chlorophyll-protein in the photosynthesis of algae and an important marine bio-active substance, from Porphyra haitanensis. The purified allophycocyanin was injected into rabbits to prepare its polyclonal antibody, and specificity of polyclonal antibody was detected by Western blotting. Tne results indicated that the purified allophycocyanin producted two bands on SDS-PAGE, which denoted the α and βsubunit of the allophycocyanin, and the purity and rate of recovery of allophycocyanin obtained were high, up to 3.5 （A650/A280） and 70.5% respectively. Tne result of indirect ELISA assay showed that the titer of the polyclonal antibody was nearly 1：100000.