CAJ | 학술논문

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pBV220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-IgG1 Fc，并克隆到pPICZαA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-IgG1 Fc融合蛋白。利用CTLL-2／IL-4R细胞测定纯化所得M5蛋白、M5-IgG1 Fc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-IgG1 Fc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（5．6×10-2 nmol／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5．8×10－2 nmol／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-IgG1 Fc融合蛋白在注射后0．5 h时血液中浓度也达到峰值，为4．7×10－2 nmol／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-IgG1 Fc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。
목적：연제보류백세포개소4수체（IL-4R）결합능력，단불구유격활하유신호활성적인백세포개소4（IL-4）돌변체M5，병통과항체Fc융합연장기체내반쇠기，위변태반응병약물연발제공기출。방법전기인합성인IL-4돌변체M5，장기극륭도pBV220표체재체，재대장간균DH5α중표체M5단백。동시구건감합기인M5-IgG1 Fc，병극륭도pPICZαA재체중，전전화당기공정필적효모GJK01，경갑순유도，분비표체M5-IgG1 Fc융합단백。이용CTLL-2／IL-4R세포측정순화소득M5단백、M5-IgG1 Fc융합단백적IL-4길항활성，최후이용ELISA시제합검측비교량자재소서체내적청제속도。결과유대장간균DH5α표체적M5단백화당기공정효모GJK01표체적M5-IgG1 Fc융합단백도구유IL-4길항활성，타문재CTLL-2／IL-4R세포상길항IL-4（5．6×10-2 nmol／ml）적EC50분별위（0．31±0．05）화（0．77±0．03）nmol／ml。소서체내M5단백재주사후0．5 h시체봉，기재혈액중적함량위5．8×10－2 nmol／ml，이재2 h시혈약농도이하강위봉치적2．8％，재8 h시저우ELISA시제합적검측한。 M5-IgG1 Fc융합단백재주사후0．5 h시혈액중농도야체도봉치，위4．7×10－2 nmol／ml，120 h시기혈약농도하강위봉치적4．3％，이168 h시저우ELISA시제합적검측한。결론 M5단백구유IL-4길항작용。유당기공정효모표체적M5-IgG1 Fc융합단백불부구유IL-4길항활성，이차재소서체내구유교장적반쇠기，위하일보장기연발위치료변태반응병적약물제공료이론기출。
Objective To develop an interleukin-4(IL-4) antagonist named M5-IgG1Fc 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 expres-sion vector pBV220 and transformed into E.coli DH5α.Chimeric gene M5-IgG1Fc obtained by overlap extension (SOE) method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZαA .Then M5-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours.The anti-IL-4 biologicial ac-tivity assay of M5 and M5-IgG1 Fc 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 M5 protein expressed in E.coli and M5-IgG1 Fc 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.M5 protein could not be detected after administration at 8 h because of the detection line . The maximum of M5-IgG1 Fc fusion protein was 4.7 ×10 -2 nmol/ml,while fusion protein M5-IgG1 Fc decreased to 4.3%of its maximum at 120 h and could not be detected at 168 h.Conclusion M5 protein has IL-4 antagonistic bioactivity .M5-IgG1 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 .