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1、生物工程学报ChinJBiotech2008,January25;24(1):153-158journals.im.ac.cnChineseJournalofBiotechnologyISSN1000-3061cjb@im.ac.cn©2008InstituteofMicrobiology,CAS&CSM,Allrightsreserved.研究简报条斑紫菜R-藻红蛋白荧光探针制备条件优化周铭,蔡春尔,柳俊秀,吴维宁,何培民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摘要:通过化学
2、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
3、、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。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OptimizationofFluorescenceProbePreparationforR-PhycoerythrininPorphyrayezoensisMingZhou,ChunerCai,JunxiuLiu,WeiningWu,andPeiminHeKeyLaboratoryofAquaticGeneticResourcesandAquacultura
4、lEcologyCertificatedbytheMinistryofAgriculture,ShanghaiFisheriesUniversity,Shanghai200090,ChinaAbstract:WeoptimizedthechemicalconjugationbetweenR-phycoerythrinandantibody.First,theR-PE(R-phycoerythrin)wasderivedwithbeterobifunctionalreagentSPDP(N-succinimid
5、yl-3-2-pyridyldithiopropionate)andantibodywasthiolatedwithDTT(dithiothreitol).Second,wedeterminedtheeffectsofthedifferentmolarratioofSPDPtoR-PEforthederivationandDTTtoIgGforthethiolation.TheresultsshowedthattheoptimummolarratioofSPDPtoR-PEforderivationwas40
6、:1,andDTTtoIgGforthiolationwas500:1.TheconjugationbetweenR-phycoerythinandantibodywasfurtherdoneforfluorescenceprobepreparation.R-phycoerythinwasconjugatedwithIgGandformedaprobecomplexbywholewavelengthscanning,electrophoresis(Native-PAGE)andfluorescencemicr
7、oscopeobservation.Keywords:R-phycoerythrin,IgG,conjugation,fluorescenceprobes,preparation,optimization藻胆蛋白是一类纯天然荧光物质,包括藻红蛋作为荧光示踪物质,正被广泛应用于免疫标记与肿[1][2]白、藻蓝蛋白、别藻蓝蛋白和和藻红蓝蛋白,它们瘤光动力治疗等领域。特别是R-藻红蛋白Received:March13,2007;Accepted:June11,2007Supportedby:theNationalH
8、ighTechnologyResearchandDevelopmentProgramofChina(863Program)(No.2006AAZ323),theMajorProgramofShanghaiEducationCommission(No.04KA01),theprojectofDengshanPlanofShanghaiScienceCommission(No.06dz12015)and
