脑囊尾蚴病脑脊液差异凝胶电泳方法的建立VIP免费

中国寄生虫学与寄生虫病杂志2008年6月第26卷第3期ChinJParasitolParasitDisJune2008,Vol.26,No.3基金项目:中国国际科技合作计划(No.2006FDA32310)作者单位:1北京热带医学研究所,首都医科大学附属北京友谊医院,北京100050;2山东省寄生虫病防治所,济宁272033*通讯作者,E-mail:ypxue@sohu.com(1BeijingTropicalMedicineResearchInstitute,BeijingFriendshipHospital,CapitalMedicalUniversity,Beijing100050,China;2ShandongInstituteofParasiticDiseases,Jining272033,China)【Abstract】ObjectiveToestablishthemethodoftwo-dimensionaldifferentialgelelectrophoresisandobtainhighresolution2Dimagesfromcerebrospinalfluid(CSF)ofpatientswithneurocysticercosis.MethodsCSFsampleswerecollectedfromfourpatientsdiagnosedasneurocysticercosisclinicallyandbyELISA,computedtomography(CT)ormagneticresonanceimaging(MRI),andfromfourhealthysubjectswithoutneurologicaldisorders.TheCSFsampleswereprecipitatedwithcoldacetone,thenpooledbyequalamountaspatientsandcontrols.Theinternalstandardcomprisedequalamountsofproteinsextractedfrombothgroups.Internalstandard,andproteinsfromthetwogroupswerelabeledpriortoelectrophoresiswithspectrallyresolvablefluorescentdyes,cyanindye2(Cy2),Cy3andCy5.Sodiumdodecylsulfonatepolyacrylamidegelchromatography(SDS-PAGE)andtwo-dimensionaldifferentialin-gelelectrophoresis(2-DDIGE)oflabeledsampleswerethenrun.ThedifferentialexpressedproteinsshowedintheimagesofSDS-PAGEand2-DDIGEgelsscannedwith488nm,532nmand633nmwavelengthlaserwereanalyzedbyImageQuantandDeCyde5.0respectively.Spotdetectionandquantificationwasperformedforthedifferentialin-gelanalysis(DIA)moduleofDeCyder.Biologicalvariationanalysis(BVA)moduleofDeCyderwasmatchedgel1andgel2imagestoprovidedataondifferentialproteinexpressionlevelsbetweenthetwogroups.ResultsTheImageQuantresultdisplayedthattheCSFproteinwascompatiblewiththedye,andthedifferenceofproteinamountwasrevealedbythedifferenceoffluorescenceintensity.DIAindicatedthattherewere896and894proteindotsongel1andgel2respectively,and90%ofthemwerematchedeachother.BVAshowedthattherewere55proteinspotswithdifferentexpressionallevelbetweenneurocysticercosisandcontrolgroups.Proteinspotswithtwo-foldincreaseordecreasewere47脑囊尾蚴病脑脊液差异凝胶电泳方法的建立【论著】文章编号:1000-7423(2008)-03-0174-05李静宜1,田小军1,黄勇2,杨艳君2,马巧荣2,薛燕萍1*【摘要】目的建立脑脊液差异凝胶电泳方法,以获得高分辨率的荧光差异双向电泳图谱。方法取确诊的4例脑囊尾蚴病患者脑脊液和4位健康人脑脊液,分别用冰丙酮沉淀法除盐提取蛋白,均等量混合后为脑囊尾蚴病组与健康人对照组,两组总蛋白等量混合为内标组。将各组蛋白分别经花青染料2(Cy2)、Cy3和Cy5标记,再进行单向十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和双向差异凝胶电泳(2-DDIGE),分别在波长488nm、532nm和633nm激发光下扫描,所呈图像分别用ImageQuant和DeCyder5.0图像分析软件进行蛋白表达差异分析。用DeCyder中的胶内差异分析(DIA)模块对2-DDIGE扫描图像进行蛋白质点检测和含量分析。用DeCyder中的生物学差异分析(BVA)模块分析两组蛋白表达的生物学差异。结果ImageQuant分析结果表明,所有脑脊液全蛋白样品均能被Cy2、Cy3和Cy5标记,荧光强度随蛋白含量的上升而增强。DIA分析显示,胶1和胶2分别检测到896和894个蛋白质点。胶2与胶1进行匹配,蛋白质点匹配率达90%。BVA分析发现,在脑囊尾蚴病组与健康人组脑脊液蛋白共有表达量变化超过两倍的差异蛋白质点55个,其中在脑囊尾蚴病组中表达上调2倍的有47个蛋白质点,下调2倍以上的有8个蛋白质点。结论以脑囊尾蚴病患者脑脊液建立的差异凝胶电泳方法,获得的2-DDIGE图谱,图像清晰,分辨率高,为脑囊尾蚴病蛋白质组学研究奠定了基础。【关键词...