| 条斑紫菜R-藻红蛋白荧光探针制备条件优化 |
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| 引用本文： | 周 铭,蔡春尔,柳俊秀,吴维宁,何培民.条斑紫菜R-藻红蛋白荧光探针制备条件优化[J].生物工程学报,2008,24(1):153-158. |
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| 作者姓名： | 周 铭 蔡春尔 柳俊秀 吴维宁 何培民 |
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| 作者单位： | 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 |
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| 基金项目： | 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)和上海市教委优势(重点)学科项目(No. Y1101)资助。 |
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| 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。
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| 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 |
| 收稿时间： | 2007-03-13 |
| 修稿时间： | 2007-06-11 |
Optimization of Fluorescence Probe Preparation for R-Phycoerythrin in Porphyra yezoensis |
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| Ming Zhou,Chuner Cai,Junxiu Liu,Weining Wu and Peimin He.Optimization of Fluorescence Probe Preparation for R-Phycoerythrin in Porphyra yezoensis[J].Chinese Journal of Biotechnology,2008,24(1):153-158. |
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| Authors: | Ming Zhou Chuner Cai Junxiu Liu Weining Wu and Peimin He |
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| Institution: | Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology Certificated by the Ministry of Agriculture, Shanghai Fisheries University, Shanghai 200090, China;Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology Certificated by the Ministry of Agriculture, Shanghai Fisheries University, Shanghai 200090, China;Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology Certificated by the Ministry of Agriculture, Shanghai Fisheries University, Shanghai 200090, China;Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology Certificated by the Ministry of Agriculture, Shanghai Fisheries University, Shanghai 200090, China;Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology Certificated by the Ministry of Agriculture, Shanghai Fisheries University, Shanghai 200090, China |
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| 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. |
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| Keywords: | R-phycoerythrin IgG conjugation fluorescence probes preparation optimization |
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