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:(1)依据 p H6抗原的生化特征及表达条件 ,对不同温度 (37℃和 2 8℃ )及不同酸碱度 (p H6和 p H7)条件下培养细菌的提取物进行了电泳图型对比 ;(2 )依照上述电泳图型 ,并结合该抗原的分子量 ,确定了 p H6抗原的电泳迁移位置 ;(3)采用 KCNS洗脱、(NH4) 2 SO4盐析、制备电泳的方法提取和纯化了鼠疫耶尔森氏菌 (Yersinia pestis) p H6抗原 ,SDS-PAGE分析表明 ,提取物均一 ,表征分子量符合 ,回收率较高 ;(4)使用纯化抗原免疫普通小鼠 5只、BAL B/ c小鼠 3只 ,免疫效果良好 ,利用获得的免疫血清 ,建立了 p H6抗原多克隆抗体的 EL ISA检测技术 ,应用于融合细胞中阳性克隆的筛检及实验动物的免疫效果评价 ,应用结果证实 ,检测敏感、有效 ;(5 )利用细胞融合技术获得杂交瘤 17株 ,对其中的 4株进行了 3次再克隆并经半年反复冷冻、复苏及上清检测 ,证实瘤株分泌抗体稳定 ;(6 )对所获单抗进行了 Western blotting(蛋白印迹试验 )鉴定 ,结果显示抗体特异。
: (1) According to the biochemical characteristics and expression conditions of p H6 antigen, the comparison of the electrophoresis patterns of the extracts of bacteria cultured under different temperature (37 ℃ and 28 ℃) and different pH (p H6 and p H7) (2) According to the above electrophoresis pattern, combined with the molecular weight of the antigen, to determine the location of electrophoresis migration p H6 antigen; (3) KCNS elution, (NH4) 2 SO4 salting out, the preparation of electrophoresis extraction and purification The Yersinia pestis p H6 antigen was analyzed by SDS-PAGE. The results showed that the extracts were homogeneous and the molecular weight was consistent and the recovery rate was high. (4) Five BALB / c mice were immunized with the purified antigen, 3 mice with good immune effect. The EL ISA detection technology of polyclonal antibody against p H6 antigen was established and used to screen positive clones in fusion cells and evaluate the immune effect of experimental animals. The results confirmed (5) Seventeen hybridomas were obtained by cell fusion technique, and four of them were re-cloned three times and were semi-frozen, resuscitated and supernatant tested for half a year to confirm the stable secretion of tumor strains. ( 6) The obtained monoclonal antibody Western blotting (Western blot test) identification, the results showed that antibodies specific.