摘要
目的：构建和表达抗人IL-2Rα基因工程抗体Fab片段，并测定该抗体片段的生物学活性。方法：采用连续PCR方法构建抗人 IL-2Rα抗体 Fab克隆载体5G1-pA22，并通过限制性酶切反应将抗人 IL-2Rα抗体 Fab基因从克隆载体 5G1-pA22重组人表达载体pET-28b，并在大肠杆菌BL21（DE3）中用IPTG诱导以包含体形式高效表达，该表达产物约占菌体蛋白20％以上。经超声破碎、洗涤、变性和复性后，采用双抗体夹心ELISA法及体外抗体竞争抑制试验鉴定表达产物的生物学活性。结果：抗人IL-2Rα基因工程抗体Fab成功构建，并以包含体形式表达，表达产物的产量达1mg/ml。双抗体夹心ELISA法试验表明抗体 Fab段与 IL-2Rα有特异的结合能力，体外抗体竞争抑制实验表明 Fab段可桔抗 IL-2与活化T细胞表面 IL-2Rα有特异的结合，抑制T细胞活化，增殖，抑制率达90％以上。结论：成功地构建了抗人IL-2Rα抗体Fh段，并获得高效表达，其具有较好的生物学活性。
Abstract Objective: To construct and express anti-IL-2Rα genetically engineering antibody Fab and identify its biological activity.Methods: PCR were used to construct cloning vector 5G1-pA22 of anti-IL-2Rα antibody Fab,by digestion of vector 5G1-pA22, anti-IL-2Rα an-tibody Fab gene fragment was obtained and then this fragment was coupled with digesting product of pET28b plasmid by EcoRI and HindIII toget a recombinant expression vector 5G1-pET28b, it was highly expressed in the E. coli BL21(DE3) in the form of inclusion bodies, compoing20% of the total protein. After the renaturation of antibody Fab, its binding activity was identified by ELISA and antibody competitive inhibitionassay in vitro. Results: Anti-IL-2Rα antibody Fab was successfully constructed and highly expressed in the form of inclusion bodies, its yield wasup to 1 mg/ml.When applied to ELISA, antibody Fab exhibited good spectfic reactivity with rIL-2R, antibody competitive inhibition assay invitro indicated that antibody Fab can compete with IL-2 for binding IL-2R in activated T cell surface.As a result,the activation and proliferationof T cells were inhibited and inhibition percentage exceeded 90%. Conclusion: Had successfully constructed and expressed anti-IL-2Rα geneti-cally engineering antibody. Fab,it had good biological ativity.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2002年第5期305-308,共4页
Chinese Journal of Immunology
基金
吉林省计委攻关课题资助项目
