抗KOD聚合酶单克隆抗体的制备(英文)  

Preparation of Monoclonal Antibodies Against KOD DNA Polymerase

作　　者：余晓岚[1] 李玉[1] 王飞[1] 钟星[1] 陈亮[1] 马立新[1] 

机构地区：[1]湖北大学生命科学学院,湖北武汉430062

出　　处：《化学与生物工程》2010年第3期63-66,共4页Chemistry & Bioengineering

基　　金：国家自然科学基金资助项目(30801008);国家863计划资助项目(2006AA02A310);湖北省自然科学基金资助项目(2008CDB110)

摘　　要：单克隆抗体具有高度的均一性和专一性。抗KOD聚合酶单克隆抗体，可以特异性结合于KOD聚合酶，抑制PCR反应前常温状态下的多聚酶活性，显著提高PCR反应的特异性。为了提高KOD聚合酶PCR反应的特异性，以KOD聚合酶为抗原，制备了两株抗KOD聚合酶的单克隆抗体。以大肠杆菌表达的重组蛋白KOD聚合酶为抗原免疫BALB／c小鼠，然后将免疫小鼠的脾细胞与SP2／0骨髓瘤细胞融合，通过HAT选择培养、ELISA检测和有限稀释法克隆，建立了2株能稳定分泌抗KOD聚合酶单克隆抗体的杂交瘤细胞，分别命名为1F5与3G6，对应的抗体亚型分别是IgGl和IgG2a。染色体计数结果分别是85～90和83～86。1F5与3G6的细胞上清和腹水的ELISA效价分别为1：2^8，1：2^9和1：2^11，1：2^11。间接免疫荧光证实制备的单克隆抗体的特异性良好。该结果为进一步建立高特异性的PCR方法奠定了良好的基础。Monoclonal antibody （McAb） was advanced in homogeneity and specificity. McAb against KOD DNA polymerase can bind with polymerase and the binding of McAb with polymerase can inhibit the activity of the KOD DNA polymerase at room temperature and improve the specificity of PCR remarkably. Two hybridoma lines producing McAb against KOD polymerase have been established. BALB/c mice was immunized with KOD polymerase expressed in E. coli, the stimulated spleenocytes were confused with SP2/0 mouse myelomas in HAT-selective culture to produce hybridomas. Two hybridoma lines specific to KOD polymerase were selected, designated 1F5, 3G6, expressed antibodies of subclasses IgG1 and IgG2a respectively. The number of chromosome of the two hybridoma lines was 85-90 and 83-86 respectively. The ELSA titers of cell supernatant and ascites of 1F5 and 3G6 were 1 ： 2^8 , 1 ： 2^9 and 1 ： 2^11 , 1 ： 2^11 respectively. By indirect immunofluorescence assay, the two McAb reacted with cells transfected with pcKOD and did not react with control cells. This result founded a base for establishing higher specificity PCR pathway.

关 键 词：KOD聚合酶 单克隆抗体 ELISA检测 制备 

分 类 号：Q511[生物学—生物化学]