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

作　　者：周铭[1] 蔡春尔[1] 柳俊秀[1] 吴维宁[1] 何培民[1] 

机构地区：[1]上海水产大学农业部水产种质资源与养殖生态重点开放实验室,上海200090

出　　处：《生物工程学报》2008年第1期153-158,共6页Chinese Journal of Biotechnology

基　　金：国家高技术研究与发展计划(863计划)项目(No.2006AAZ323);上海市教委重点项目(No.04KA01);上海市科委登山计划项目(No:06dz12015);上海市教委优势(重点)学科项目(No.Y1101)资助~~

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。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.

关 键 词：R-藻红蛋白.单克隆抗体 交联 荧光探针 制备 条件优化 

分 类 号：Q946[生物学—植物学]