重组水蛭素的聚乙二醇修饰.doc

重组水蛭素的聚乙二醇修饰李雪芹，候蓓蓓，赵军，田明玉，修志龙*大连理工大学环境与生命学院大连116024摘要：目的水蛭素因血浆半衰期短而严重限制了其临床应用，聚乙二醇修饰能有效地延长其半衰期。本文通过比较不同修饰位点、修饰方法所得单修饰产物的比率、纯度及其活性保留率，从而得到修饰专一性强且活性保留率较高的修饰策略。方法采用液相和固相修饰方法,用琥珀酰亚胺活化的PEG20kDa分别在pH6.0、8.0的条件下对水蛭素的His和Lys进行定点修饰；用SDS-PAGE分析产物的修饰度，并用离子交换柱对修饰后的产物进行分离，然后用凝血酶滴定法测定水蛭素单修饰产物的体外抗凝活性。另外用分子动力学模拟方法预测了pH8.0条件下PEG修饰的位点。结果在pH6.0、8.0的条件下，水蛭素单修饰率都高达90%以上，但二者的单修饰活性保留率相差较大。在pH6.0时，液、固相单修饰活性保留率为34%、34.8%；而在pH8.0时分别为55%、96%。结论在pH8.0条件下，采用“离子交换柱辅助”固相修饰的方法对Lys进行定点修饰能得到较高产率和较高活性保留率的单修饰产物。关键词：重组水蛭素；SC-mPEG；抗凝活力；分子动力学模拟PEGylationofRecombinantHirudinLiXueqin，HouBeibei，ZhaoJun，TianMingyu，XiuZhilong*DepartmentofBioscienceandBiotechnology,SchoolofEnvironmentalandBiologicalScienceandTechnology,DalianUniversityofTechnology,Dalian116024,ChinaAbstract:OBJECTHirudinisthemostpotentinhibitorofthrombinfoundinnature.Althoughhirudinhasthestrongestanti-thrombinactivityinvivo,itsshorthalf-lifeinserumsignificantlylimitsitsclinicalanticoagulantapplication.Currently,PEGylationiscommonlyusedasaneffectivemethodtoprolongitshalf-lifeinserum.OurobjectofexperimentistochoosethebestPEGylationmethodaccordingtothemonoPEGylatedrecombinanthirudin’spurityandtheanticoagulantactivityinvitro.METHODSSolutionmethodandsolidmethodassistedby“packed-bed”andanionexchangecolumnwereusedtofavortheformationofmono-PEGylatedhirudin.ThemildacidicandmildalkalinePEGylationstrategywasusedtotargetHisresidueandLysresidueofhirudinrespectivelyusingSC-mPEG20kDa.Thepegylatedproductswereisolatedbyanionexchangechromatogram.SDS-PAGEwasusedtoanalyzethepurityofPEGylatedhirudin.ThrombinmethodwasusedtoanalyzetheanticoagulantactivityofPEGylatedhirudin.MoleculardynamicsmodelingwasusedtojudgetheprobabilityofPEGylationsite.RESULTSThemono-PEGylatedproductwithapurityofmorethan90%wasobtainedatpH6.0andpH8.0.Butalargediffe