人源化抗p185单链抗体/人白细胞介素-2双功能融合蛋白在293细胞中的高效表达

High efficient expression of a humanized single-chain Fv antibody/human interleukin-2 fusion protein directed against p185 in 293 cell line

目的 在 2 93细胞中表达人源化单链抗体 /人白细胞介素 2 (hIL 2 )双功能融合蛋白及评价其生物学活性。方法 将构建的表达载体转染 2 93细胞 ,G418筛选阳性克隆建立稳定表达细胞系 ,免疫印迹法 (Westernblot)、酶联免疫吸附试验 (ELISA )及 [3 H]脱氧胸苷 (3 H TdR)渗入法检测细胞上清中融合蛋白浓度和生物学活性。结果 融合蛋白浓度达 (10 2 .0± 4.2 ) g/L ,5 μl细胞上清即可在 45× 10 3 处呈现清晰蛋白条带。其中hIL 2无活性丢失 ,与标准对照比较差异无显著性 (P >0 .0 5 ) ;抗体的抗原结合活性有部分下降 ,与标准对照比较差异有非常显著性 (P <0 .0 1)。结论 该融合蛋白可在 2 93细胞系中得到高效表达 ,其中hIL 2部分无活性丢失 。

Objective To express a bispecific humanized single-chain Fv antibody/human IL-2 fusion protein against p185 in 293 cell line and verify its bioactivities.Methods The constructed expression vectors were transfected into 293 cells.The positive clone was obtained to make stable cell line with G418 selection.Western Blot,ELISA and 3H-TdR were used to measure the concentration of the fusion protein and its bioactivities in the supernatant.Results The concentration of fusion protein was (102.0±4.2) mg/L in supernatant,and only 5 μl of the supernatant was able to be shown to migrate as single band of 45×10 3 by Western blot analysis.The fusion protein was determined to retain the bioactivities of hIL-2 (P>0.05 vs standard rhIL-2).The loss of antigen-binding activity was shown to be less two orders of magnitude than that of 520C9-rhIL-2 chemical conjugate (P<0.01).Conclusion The fusion protein can be highly expressed in 293 cell line.The protein retained its hIL-2 bioactivities and acceptable antigen-binding specificity.