作　　者： ; ; ; ; ; ; ;

机构地区： 中国农业科学院兰州畜牧与兽药研究所

出　　处： 《黑龙江畜牧兽医》 2013年第2期75-78,166,共5页

摘　　要： 为了优化抗喹乙醇单克隆抗体制备中聚乙二醇(PEG)诱导细胞融合的最适条件,试验采用流式细胞仪确定SP2/0细胞的对数生长期,以融合率和阳性率为评价指标,分析血清加入量、500 mL/LPEG-1000溶液方法及反应时间和次黄嘌呤、氨基蝶呤、胸腺嘧啶脱氧核苷(HAT)加入时间对SP2/0细胞与脾细胞融合效果的影响。结果表明:150,200 mL/L胎牛血清融合率分别为86.5%、88.5%;生理盐水和无血清DMEM配制的500 mL/L PEG-1000融合率和阳性率分别为61.4%、3.1%,30.2%、1.0%;PEG-1000反应1.75,3.00 min融合率和阳性率分别为61.4%、3.1%,74.5%、6.3%;融合后培养0,24 h加入HAT,结果融合率和阳性率分别为95.8%、17.7%,94.3%、26.0%。说明150 mL/L胎牛血清、500 mL/L PEG-1000生理盐水、反应3.00 min、培养24 h后加入HAT时,细胞融合率和阳性率较高,是抗喹乙醇单克隆抗体制备中PEG诱导细胞融合的最适条件。 To optimize the conditions of PEG - induced cells fusion in the preparation of monoclonal antibodies against olaquindox, the logarith- mic growth stage of SP2/0 cells was determined with flow cytometry. The effect of the four factors on the cell fusion between the SP2/0 cells and spleen cells was analyzed, by comparing the cell fusion rate and the positive rate, the four factors included fetal bovine serum concentration, the preparation and reaction time of 500 mL/L PEG- 1000 and the addition time of hypoxanthine,aminopterin, and thymidine（HAT）. The results showed that the fusion rates treated with 150 mL/L and 200 mL/L fetal bovine serum （FBS） were 86.5% and 88.5%, respectively. The fu- sion rates treated with normal saline and 500 mL/L PEG - 1000 with serum - free DMEM were 61.4% and 30.2%, and the positive rates trea- ted with normal saline and 500 mL/L PEG - 1000 with serum - free DMEMwere 3.1% and 1.0%, respectively. The fusion rate and positive rate were 61.4% and 3.1% after PEG reacting for 1.75 rain, and 74.5 % and 6.3 % after PEG reacting for 3.00 min, respectively. The fu- sion rate and positive rate were 95.8% and 17.7% with HAT being added to the culture medium at 0 h after cell fusion, respectively. They were 94.3% and 26.0% with HAT being added at 24 h after cell fusion. In the experiment, the results indicated that the optimal conditions of PEG -induced cells fusion in the preparation of monoclonal antibodies against o｜aquindox, were 150 mL/L FBS, 500 mL/L PEG - 1000 in normal saline, the reaction time for 3.00 min, and the addition of HAT to the culture medium at 24 h after the culture.

关 键 词： 喹乙醇 单克隆抗体 细胞融合 聚乙二醇 优化

领　　域： [农业科学] [农业科学] [农业科学]