基于固定化酶的蛋白质磷酸酶抑制率法测定腹泻性贝类毒素

Research on detection of Diarrhetic Shellfish Poisoning by using immobilized protein phosphatase inhibition assay

中文关键词:  腹泻性贝类毒素  蛋白质磷酸酶  快速检测  酶抑制率法

英文关键词:Diarrhetic Shellfish Poisoning  Protein phosphatase 2A  Rapid detection  Enzyme inhibition assay

中文摘要:

      目的 建立和优化用于检测腹泻性贝类毒素(DSP)的固定化蛋白质磷酸酶2A(PP2A)抑制率法。方法 以分离提取的PP2A为材料,根据DSP抑制PP2A酶活性的原理,建立用于检测DSP的固定化PP2A酶抑制率法,并对最佳酶用量和反应体系参数等进行了优化。结果 最佳的固定化PP2A酶蛋白量2μg、固定化配比为琼脂糖与PP2A酶蛋白量为1:1、pH值为8.4；采取4参数Logistic曲线法进行标准曲线拟合,曲线R2值达0.994,检测限为3.45μg/L,实际样品测定时具有良好的准确性和重复性。结论 建立的固定化PP2A酶抑制率法有望作为低成本、操作简单的DSP快速筛检方法推广应用。

英文摘要:

      Objective The aim of the present study was to develop and optimize an immobilized enzyme inhibition assay for Diarrhetic Shellfish Poisoning (DSP). Methods Based the principle which DSP could inhibit the activity of protein phosphatase 2A (PP2A), the assay was established by using the extracted PP2A in the present study. The parameters such as the amount of enzyme and reaction condition were optimized. Results The optimal PP2A immobilizedSenzymeSprotein wasS2μg, the optimal ratioSfor agaroseSandSPP2A enzyme proteinSwas 1:1, andSthe optimal pH value was 8.4. The standard curve fitting was made by the 4-parameter Logistic curve method. R2 value of curve fitting was 0.994, which suggested standard curve fitting has good fit. The detection limit of OA was 3.45μg/L. This assay for detecting the actual sample has good accuracy and repeatability. Conclusion These results suggested that the immobilized enzyme inhibition assay may be used a low-cost and easy operation method for the rapid detection of DSP.