磷酰化组氨酸特异性抗体的制备与应用

Preparation and Application of Specificity Phosphohistidine Antibody

以4-碘吡唑和亚磷酸二乙酯为初始原料,经3步反应合成了新型的磷酰化组氨酸结构类似物,并以其为半抗原制备了磷酰化组氨酸特异性抗体(Anti-pHis).采用酶联免疫吸附(ELISA)、斑点印迹(Dot blot)和免疫印迹(Western blot)实验等方法对Anti-pHis的特异性进行了检测.结果表明,Anti-pHis对组氨酸磷酰化蛋白表现出优异的亲和力和选择性识别能力,并可应用于微生物细胞裂解液中组氨酸磷酰化蛋白的检测.

A novel phosphorylated histidine analogue was synthesized using 4-iodopyrazole and diethyl phosphite as raw materials and characterized by nuclear magnetic resonance spectroscopy(NMR).It was used as hapten to prepare phosphohistidine antibody(Anti-pHis).Enzyme-linked immunosorbent assay,dot blot and western blot methods were used to detect the specificity and sensitivity of the Anti-pHis.The results showed that the Anti-pHis had excellent avidity and sensitivity for histidine phosphorylated protein,and the Anti-pHis was successfully applied to the detection of histidine phosphorylated protein in the lysate of microbial cells.