长效人白细胞介素4拮抗剂的研究

Interleukin-4 antagonists with longer half-life in plasma

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 an interleukin-4 （IL-4） antagonist named M5-IgG1 Fc protein constructed by genetic engineering of antibody Fc fragment-cytokine mutein fusion protein which has a long half-life time in plasma. M5-IgG1 Fc protein binds to IL-4 receptor but cannot activate downstream signalling pathway, which provides a basis for drug develop- ment for allergic diseases. Methods The synthesized interleukin-4 mutant gene （ named M5 ） was cloned into the expression vector pBV220 and transformed into E. coli DH5 ct. Chimeric gene M5 -IgG1 Fc obtained by overlap extension （SOE） method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZaA . Then Ms-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours. The anti-IL-4 biologicial activity assay of M5 and M5-IgG1Fc was performed with CTLL-2/IL-4R cells and detected with MTT colormetry. Finally, the half-life time of M5 and M5-IgG1 Fc protein in mice was compared by detecting the remaining amount in plasma with ELISA kit. Results The Ms protein expressed in E. coli and Ms -IgG1Fc fusion protein expressed in P. pastoris GJK01 both had IL- 4 antagonistic bioactivity. The EC50 of both, which inhibited 5.6 ×10^-2 nmol/ml of IL-4, were 0.31 ±0.05 and 0.77 ± 0.03 nmoL/ml, respectively. The maximum of M5 in plasma at 0.5 h was 5.8 ×10^-2 nmol/ml but the remaining amount was 2.8% of the maximum at 2 h. Ms protein could not be detected after administration at 8 h because of the detection line. The maximum of Ms-IgG1 Fc fusion protein was 4.7 ×10^-2nmol/ml, while fusion protein M5 -IgG1Fc decreased to 4.3% of its maximum at 120 h and could not be detected at 168 h. Conclusion Msprotein has IL-4 antagonistic bioactivity. M： IgGl Fc fusion protein expressed in glycoengineered P. pastoris GJK01 has IL-4 antagonistic bioactivity and long retention time in mice ,which can be potentially used for treatment of allergic diseases.