Preparation and optimization of human anti-Trop-2 antibody fragment Fab
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摘要:
目的:探讨人源抗Trop-2抗体片段Fab在大肠杆菌中的诱导表达,优化蛋白表达及纯化的条件-方法:将含有抗Trop-2 Fab 抗体基因的质粒转化宿主菌E.coli TOP10F ′大肠杆菌,比较诱导前培养液更换对抗Trop-2 Fab片段表达量的影响;比较不同诱导温度-诱导时间及诱导前加入葡萄糖对蛋白表达的影响;大量表达的抗Trop-2 Fab经亲和层析纯化后,用免疫荧光和流式细胞术检测其免疫学特性-结果:在培养液中加入5 g/L的葡萄糖,人源抗Trop-2抗体Fab片段的表达产量明显增加;16℃的诱导温度比其它温度能表达更多的Fab分子;加诱导剂12 h,目的蛋白的表达量至峰值-结论:本实验结果为在原核系统中大量制备抗Trop-2抗体片段及后续的肿瘤靶向治疗提供了基础-
Abstract:
Objective: To explore the induced expression of human anti-Trop-2 antibody fragment Fab in E.coli TOP10F ′ and to optimize the expression and purification conditions of protein. Methods: The plasmid containing anti-Trop-2 Fab antibody gene was transformed into E.coli TOP10F ′. It was tested to replace the culture medium before induction of anti-Trop-2 Fab expression. The effects were compared in different induction temperatures,induction times and glucose added before induction on protein expression. Anti-Trop-2 Fab was purified by affinity chromatography and its immunological characteristics were evaluated by immunofluorescence and flow cytometry. Results: In the culture medium by adding 5 g/L of glucose,the expression of anti-Trop-2 Fab significantly increased. The temperature of 16℃ can induce more expression of Fab than other temperatures. The expression of anti-Trop-2 Fab reached peak after adding inducer for 12 hours. Conclusion: This study provides the basis of large-scale preparations of anti-Trop-2 Fab in prokaryotic expression system and subsequent utilization of anti-Trop-2 Fab in tumor targeted therapy.
