条斑紫菜R-藻红蛋白荧光探针制备条件优化

Optimization of Fluorescence Probe Preparation for R-Phycoerythrin in Porphyra yezoensis

摘要:

通过化学方法使条斑紫菜R-藻红蛋白与抗体进行交联以制备荧光探针, 并对制备条件进行优化。首先采用异双功能试剂SPDP (N-琥珀酰亚氨基-3-2-吡啶基二硫丙酸醇)和DTT（二硫苏糖醇）分别使 R-PE(R-藻红蛋白)衍生化、IgG（单克隆抗体）巯基化, 其次测定了SPDP与R-PE不同摩尔比对R-PE衍生化的影响、DTT与IgG不同摩尔比对IgG巯基化的影响, 结果表明：SPDP与R-PE的最佳摩尔比为40：1, DTT与IgG的最佳摩尔比为500:1。在此基础上, 建立了R-PE与IgG交联的制备技术, 并应用全波长扫描吸收光谱、电泳分析和荧光显微镜观察等监测和分析技术, 证实了藻红蛋白与抗体已成功交联形成了复合物。

Abstract:

We optimized the chemical conjugation between R-phycoerythrin and antibody. First, the R-PE (R-phycoerythrin) was derived with beterobifunctional reagent SPDP(N-succinimidyl-3-2-pyridyldithio propionate) and antibody was thiolated with DTT(dithiothreitol). Second, we determined the effects of the different molar ratio of SPDP to R-PE for the derivation and DTT to IgG for the thiolation. The results showed that the optimum molar ratio of SPDP to R-PE for derivation was 40:1, and DTT to IgG for thiolation was 500:1. The conjugation between R-phycoerythin and antibody was further done for fluorescence probe preparation. R-phycoerythin was conjugated with IgG and formed a probe complex by whole wavelength scanning, electrophoresis (Native-PAGE) and fluorescence microscope observation.