目的探索快速有效的从稳定转染后的悬浮多克隆细胞中分离、纯化并鉴定出单克隆的技术改良法。方法电穿孔法稳定转染后的T淋巴细胞系先在分离培养基中扩增成多克隆,用有限稀释法分离出单个细胞,一部分用丝裂霉素C处理的饲养细胞培养扩增单克隆,一部分进行无饲养细胞扩增,用未转染的T淋巴细胞系的新鲜培养上清换液,对比两法的单克隆得率。检测单克隆的靶基因整合则先用荧光素酶报告实验再结合引物PCR扩增法,筛选出表达标记蛋白并整合有完整特异基因片段的单克隆。结果两种方法在分离培养基中存活增殖的活细胞克隆数量比例分别为5.27%和4.55%,无显著差异,通过荧光素酶报告基因实验确定完整整合外源基因的靶克隆比率(0.80%和0.57%)也无显著差异。单克隆的报告荧光素酶表达强度显著高于多克隆和对照,比较直观快速。结论无饲养细胞法避免了饲养细胞复燃污染单克隆细胞,简化了步骤提高了效率,能保证单个细胞在选择培养基中成功扩增出单克隆。荧光素酶报告基因实验能快速、有效检测出外源整合基因的表达。 Objective To explore a rapid and effective technique for the isolation, purification and identification of monoclonal antibodies from stably transfected polyclonal cells. Methods T-lymphocytes stably transfected by electroporation were first amplified into polyclonal in isolation medium, single cells were isolated by limiting dilution method, and a portion of feeder cells treated with mitomycin C were cultured to amplify monoclonal , Part of the feeder-free cell expansion, using untransfected T lymphocyte line fresh culture supernatant fluid, compared with the two methods of the monoclonal yield. The target genes for the detection of monoclonal clones were first screened by a luciferase reporter assay combined with a primer PCR method to screen out the clones expressing the marker protein and integrating the complete and specific gene fragments. Results The number of viable cells that survived in the two methods were 5.27% and 4.55%, respectively. There was no significant difference between the two methods. The ratio of target clones (0.80% and 0.57%) also no significant difference. Monoclonal luciferase expression was significantly higher than the polyclonal and control, more intuitive and fast. Conclusion The feeder-free method avoids the re-burn of the feeder cells to pollute the monoclonal cells, simplifies the steps to improve the efficiency and ensures that the single cell can successfully amplify the monoclonal antibody in the selection medium. Luciferase reporter assay can rapidly and effectively detect the expression of foreign genes.