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为深入研究转基因玉米所表达的植酸酶蛋白的酶学性质,评价重组蛋白的致敏性和饲用安全性,必须获得高纯度植酸酶蛋白。利用识别4个不同表位的单克隆抗体制备了植酸酶蛋白亲和层析体系。结果显示,通过80%硫酸铵沉淀植酸酶蛋白粗提液初步浓缩目的蛋白后,再经过透析去除高浓度盐离子,进而通过免疫亲和层析可获得在SDS-PAGE胶上条带单一的植酸酶蛋白,比活可达470.99 U/mg。同时,将上述免疫亲和层析法对植酸酶的纯化效果与离子交换层析方法进行了对比,结果表明免疫亲和层析法具有稳定、快速和纯化产物比活高等优势,所获得的目的蛋白满足各种检测要求,优于离子交换层析方法。
In order to further study the enzymatic properties of phytase proteins expressed in transgenic maize and to evaluate the sensitization and feed safety of recombinant proteins, it is necessary to obtain high-purity phytase proteins. Phytase protein affinity chromatography was prepared using monoclonal antibodies that recognize four different epitopes. The results showed that after 80% ammonium sulfate precipitation phytase protein crude extract initial concentration of the target protein, and then by dialysis to remove high concentrations of salt ions, and then by immunoaffinity chromatography can be obtained on SDS-PAGE gel with a single band Phytase protein, specific activity up to 470.99 U / mg. At the same time, comparing the purification effect of phytase and ion exchange chromatography, the results of immunoaffinity chromatography showed that immunoaffinity chromatography has the advantage of high specific activity, stable, rapid and purified product. The target protein meets various testing requirements and is superior to ion exchange chromatography.