抗人CDC7单克隆抗体的制备与鉴定.pdf

《抗人CDC7单克隆抗体的制备与鉴定.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、2012年l2月第l9卷第35期·论著·抗人CDC7单克隆抗体的制备与鉴定李志广张现伟蒋小梅旷兴林魏大鹏章崇杰四川大学华西基础医学与法医学院免疫学教研室，四川成都610041【摘要】目的制备抗人CDC7蛋白的单克隆抗体并鉴定其生物学特性。方法以GST—CDC7为免疫原，免疫Balb／c小鼠，取其脾细胞与小鼠骨髓瘤细胞(SP2／0细胞)融合，经多次筛选及克隆化，建立可以稳定分泌抗人CDC7单克隆抗体的杂交瘤细胞株。用间接ELISA，WesternBlot，免疫细胞化学(ICC)对此抗体特性进行鉴定。结果筛选到1株能稳定分泌抗人CDC7的细胞株1F7D2

2、C9。亚类鉴定为IgG2a．轻链为kappa链，ELISA法鉴定该抗体腹水效价为1：102400，抗体亲和常数为1．558xl09L／mol，WesternBlotting分析表明，此抗体能特异识别细胞株的天然CDC7蛋白质。免疫细胞化学进一步显示CDC7分布于细胞核中。结论成功制备了抗人CDC7单克隆抗体，为研究CDC7与细胞癌变的关系建立了基础。【关键词l细胞分裂周期蛋白激酶7；单克隆抗体；DNA复制；制备；鉴定【中图分类号】R392【文献标识码】A【文章编号】1674—4721(2012)12(b)一0013—03Preparationandi

3、dentificationofamonoclonalantibodyagainsthumanCDC7LIZhiguangZHANGXianweiJIANGXiaomeiKUANGXinglinWElDapengZHANGChongfie‘DepartmentofImmunology，SchoolofPreelinicalandForensicMedicine，SichuanUniversity，Chengdu610041，China[Abstract]ObjectiveToprepareandidentifythemonoclonalantibody

4、againsthumanCDC7．MethodsBalb／cmicewereimmunizedwiththeimmunogenGST-CDC7．ThespleencellsoftheimmunizedmicewereisolatedandfusedwithSP2／0cells．Afterseveralroundsofdetectingandcloning，ahybridomacelllinesecretinganti-humanCDC7MAbwasobtained．ItsspecificitywasevaluatedwithindirectELISA

5、，WesternBlotandimmunocytochemistry．Thetitter，immunoglobulinsub-typeandaffinityconstantoftheMAhweremeasured．ResultsOnecellofhybridomanamed1F7D2C9wasobtained，whichwasidentifiedthattheheavyandlightchainsofanti-humanCDC7MAbwereIgG2aandkappa，respectively，withhighafinityof1．558x10~IJ

6、molandhightitterof1：102400．WesternblotdemonstratedthattheMAbcouldspecificallyrecog-nizethenaturalcelllysisprotein．TheimmunocytochemistryprovedthattheMAbcouldrecognizehumanCDC7expressedinthenucleiofHCCLM3cellline．ConclusionThesuccessinanti-CDC7antibodypreparationprovidesthefound

7、ationontherelationshipbetweenhumanCDC7andcancer．【Keywords】Celldivisioncycle7relatedproteinkinase；Monoclonalantibody；DNAreplication；Preparation；Identification细胞分裂周期蛋白激酶7(CDC7)是促进DNA合成的重】】．2实验材料小鼠骨髓瘤细胞株(SP2，0)购买于上海中科要激酶。在真核生物中，CDC7由574个氨基酸组成并且稳定院细胞库。HCT细胞株和HCCLM3细胞株由本实验室提供。表达。分子量

8、约64KDa，主要位于细胞核。CDC7激酶在细胞GST—CDC7由四川大学刘聪实验室提供。福氏完全佐剂、福周