人IL-2Rα基因在CHO细胞中的稳定整合和表达及其应用

Studies on the Stable Integration and Expression of IL-2Rα Gene in CHO Cells and It′s Applications

目的 应用本组制备的白细胞介素 2受体 (IL- 2 R) ,建立一种既安全又简便 ,并能定量检测白细胞介素2 (IL- 2 )生物活性的方法。方法 采用 Southern印迹杂交方法 ,检测重组分泌型 IL- 2 Rα亚基 (rs IL- 2 Rα)基因在高效表达的转染细胞基因组中的稳定整合 ;以 Western免疫印迹法测定 rs IL- 2 Rα的相对分子质量 ,建立定量检测人 IL- 2生物活性的受体与抗体夹心酶联免疫吸附测定 (r EL ISA)方法。结果 (1) Southern印迹分析表明 ,rs IL- 2 Rα基因已稳定整合到高表达的转染 CHO细胞基因组中 ;(2 ) rs IL- 2 Rα的相对分子质量为 40 0 0 0左右 ;(3)应用建立的r EL ISA方法测定人 IL- 2标准溶液结果表明 ,IL- 2的标准曲线范围为 31.2 5～ 5 0 0 .0 0 U (r=0 .9995 ) ;预先将山羊抗人 IL- 2 Ig G与 IL- 2保温后 ,所得标准曲线的斜率明显降低 (P<0 .0 5 )。结论 与传统测定 IL- 2的方法相比 ,用rs IL- 2 Rα建立的受体 -抗体夹心 r EL ISA方法不仅具有准确、特异性强及操作简便的特点 ,而且 ,所得结果直接反映了被测样品中具有结合 IL- 2 R活性的 IL-

Objectives To establish a novel bioassay method for quantitative analysis of human IL2 based on the specific binding of Interleukin2 receptor α subunit (IL2Rα) with IL2. Methods Southern blot hybridization was first used to detect the stability of integration of recombinant secretive IL2Rα (rsIL2Rα) gene into the genome of highly expressed cell line reported elsewhere; the apparent Mr of the rsIL2Rα was then determined by using Western blotting; finally, a receptorantibody sandwich ELISA method has been established for quantitative analysis of IL2. Results (1) Stable integration of rsIL2Rα gene into the genome of # 17 CHO cell line has been identified; (2) the apparent Mr of rsIL2Rα was approximately 40 000 ; (3) linear range of the standard curve obtained from the receptorbased ELISA fell between 3125 ~500 U of IL2 (r=09995). The slop of the standard curve decreased significantly when IL2 was preincubated with goat antiIL2 antibody IgG (P<001). Conclusions An IL2Rαbased IL2 ELISA has been established for laboratory and clinical use with advantages of accuracy, specificity and simplicity over other conventional bioassays for IL2 detection.