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目的研制保留白细胞介素4受体(IL-4R)结合能力,但不具有激活下游信号活性的人白细胞介素4(IL-4)突变体M5,并通过抗体Fc融合延长其体内半衰期,为变态反应病药物研发提供基础。方法全基因合成人IL-4突变体M5,将其克隆到p BV220表达载体,在大肠杆菌DH5α中表达M5蛋白。同时构建嵌合基因M5-Ig G1Fc,并克隆到p PICZαA载体中,电转化糖基工程毕赤酵母GJK01,经甲醇诱导,分泌表达M5-Ig G1Fc融合蛋白。利用CTLL-2/IL-4R细胞测定纯化所得M5蛋白、M5-Ig G1Fc融合蛋白的IL-4拮抗活性,最后利用ELISA试剂盒检测比较两者在小鼠体内的清除速度。结果由大肠杆菌DH5α表达的M5蛋白和糖基工程酵母GJK01表达的M5-Ig G1Fc融合蛋白都具有IL-4拮抗活性,它们在CTLL-2/IL-4R细胞上拮抗IL-4(5.6×10-2nmol/ml)的EC50分别为(0.31±0.05)和(0.77±0.03)nmol/ml。小鼠体内M5蛋白在注射后0.5 h时达峰,其在血液中的含量为5.8×10-2nmol/ml,而在2 h时血药浓度已下降为峰值的2.8%,在8 h时低于ELISA试剂盒的检测限。M5-Ig G1Fc融合蛋白在注射后0.5 h时血液中浓度也达到峰值,为4.7×10-2nmol/ml,120 h时其血药浓度下降为峰值的4.3%,而168 h时低于ELISA试剂盒的检测限。结论 M5蛋白具有IL-4拮抗作用。由糖基工程酵母表达的M5-Ig G1Fc融合蛋白不仅具有IL-4拮抗活性,而且在小鼠体内具有较长的半衰期,为下一步将其研发为治疗变态反应病的药物提供了理论基础。
OBJECTIVE: To develop human interleukin 4 (IL-4) mutant M5 that retains interleukin 4 receptor (IL-4R) binding but not downstream signaling activity and to prolong its in vivo half-life by Fc fusion Allergen drug development to provide the basis. Methods Human IL-4 mutant M5 was synthesized and cloned into p BV220 expression vector. M5 protein was expressed in E. coli DH5α. At the same time, the chimeric gene M5-Ig G1Fc was constructed and cloned into p PICZαA vector. The glycogen engineered yeast Pichia pastoris GJK01 was electroposed and the M5-Ig G1Fc fusion protein was induced by methanol. IL-4 antagonistic activity of the purified M5 protein and M5-Ig G1Fc fusion protein was determined by CTLL-2 / IL-4R cells. Finally, the clearance rate of the two in mice was detected by ELISA kit. As a result, the M5 protein expressed by Escherichia coli DH5α and the M5-Ig G1Fc fusion protein expressed by the glycoengineered yeast GJK01 all had IL-4 antagonistic activity and antagonized IL-4 (5.6 × 10 6) on CTLL-2 / IL-4R cells -2 nmol / ml) were (0.31 ± 0.05) and (0.77 ± 0.03) nmol / ml, respectively. M5 protein in mice reached peak at 0.5 h after injection, and its content in blood was 5.8 × 10-2 nmol / ml, while at 2 h, the concentration of M5 protein dropped to 2.8% of peak value, and was lower at 8 h Limit of detection in ELISA kits. The concentration of M5-Ig G1Fc fusion protein peaked at 0.5 h after injection, which was 4.7 × 10-2 nmol / ml. The plasma concentration of M5-Ig G1Fc fusion protein decreased to 4.3% of peak value at 120 h, but lower than that of ELISA reagent at 168 h Detection limit of the box. Conclusion M5 protein has IL-4 antagonism. The M5-Ig G1Fc fusion protein expressed by the glycoengineered yeast not only has IL-4 antagonistic activity, but also has a longer half-life in mice, which provides a theoretical basis for the next step of developing it as a drug for the treatment of allergic diseases.