抗新生血管蛋白KBP多克隆抗体的制备及鉴定.pdf

《抗新生血管蛋白KBP多克隆抗体的制备及鉴定.pdf》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、解剖学研究2011年第33卷第2期AnatRes，2011，Vo1．33．No．2105·论著抗新生血管蛋白KBP多克隆抗体的制备及鉴定陈易斐，黄莉钧，李磊，戴智育，程锐，杨霞，高国全(中山大学中山医学院生物化学教研室，广东广州510080)【摘要】目的激肽释放酶结合蛋白(Kallikrein—bindingprotein，KBP)具有抑制血管新生的高活性。但目前尚无KBP的特异性抗体，限制其进一步研究。本研究拟制备高灵敏度和高特异性的KBP抗体并对其进行初步评估。方法构建含大鼠源性KBP序列的pET．28a重组载体

2、的大肠杆菌BL21(DE3)，在此基础上，扩增表达菌，经异丙基B．D一硫代半乳糖苷(IPTG)诱导表达，Ni．NTA色谱柱亲和层析纯化获得KBP重组蛋白。利用SDSPAGE和Westernblot方法对KBP进行鉴定。以纯化产物为抗原免疫新西兰大白兔，获得兔抗KBP血清。亲和纯化获得多克隆抗体。应用Elisa和Western．blot方法检测纯化抗体的效价和特异性。结果KBP融合蛋白得到高表达，且纯度大于85％。用该融合蛋白免疫大白兔后得到的抗KBP抗体，效价达1：512000，并特异性识别大鼠和人组织中天然的KBP

3、蛋白。结论以纯化的KBP重组蛋白为抗原，获得KBP多克隆抗体，具有较好的效价和特异性，为KBP功能和作用机制的深入研究提供了必要的工具。【关键词】KBP重组蛋白；多克隆抗体制备；抗体评价Preparationandpurificationofrabbitanti-KBPpolyclonalantibodyCHENYi-fei，HUANGLi-jun，LILei，DAIZhi—yu，CHENGRui，YANGXia，GA0Guo—quart．DepartmentofBiochemistry&MolecularBiolo

4、gy，ZhongshanMedicalSchool，SUNYat—senUniversity，Guangzhou510080，China【Abstract】ObjectiveKBP，shortfromkallikreinbindingprotein，isaendogenousproteinwithhighantiangiogenicpotentia1．ThefurtherstudyaboutKBPiSrestrictedbecausethereiSstillnotspecificanti．KBPantibody．Th

5、isstudyiSdesignedtoobtainhighlysensitiveandspecificanti—KBPpolyclonalantibodyandealTyontheprimaryevaluationofit．MethodsTheexpressionvectorpET28a—KBPhadbeenreconstructedandtransformedintoBL21(DE3)byOUrlab．RecombinantKBPproteinwasinducedtoexpressbyIPTGandpurified

6、byNi—NTAHisBindresin．PurifiedKBPwasidentifiedbySDS-PAGEandwesternblotanalysisandusedtoimmunizetheNewZealandrabbits．Theanti—KBPpolyclonalantibodyispreparedbyafinitypurificationfromtherabbits’serumafterimmunization．ThetiteroftheantibodywasdetectedbyELISAanditsspe

7、cificitywasanalyzedbyWesternblot．ResultsTheKBPrecombinantproteinwashighlyexpressedinE．coilandpurityofpurifiedproteinismorethan85％．Thetiterofrabbitanti．KBPpolyelonalantibodyobtainedafterimmunizationandpurificationiSabout1：512．000．Andtheanti—KBPantibodyindentifie

8、snativeKBPofratandhumantissuesspecifically．ConclusionHighlyspecialpolyclonalantibodyforKBPwassuccessfullygenerated，whichisessentialforfurtherstudyofthefunctionandmechanismso