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目的:将快速蛋白液相色谱系统(FPLC)与阴离子交换层析相结合,探索在大肠杆菌(E.coli)分泌表达的人抗地高辛(Dig)单链抗体(ADAscFv)的纯化条件。方法:①通过培养E.coliHB2151将人ADAscFv分泌表达至培养基中,离心制备培养基上清;②加入硫酸铵,收集其饱和度为0~50%的组分即为初步纯化的人ADAscFv;③将阴离子交换色谱柱与FPLC相连接后上样,分别采用含NaCl的Tris-HCl缓冲液进行线性浓度梯度洗脱及阶梯浓度梯度洗脱,紫外分光光度计自动检测洗脱峰;④通过ELISA及SDS-PAGE分别分析回收样品中人ADAscFv的抗原结合活性及纯度。结果:①2种洗脱方法均有6个洗脱峰;②ELISA显示,第2个洗脱峰回收样品中的抗原结合活性最高;③SDS-PAGE显示,第2个洗脱峰回收样品呈现与人ADAscFv分子量相符的单一条带。结论:该方法能有效地纯化在E.coli分泌表达的人ADAscFv。
OBJECTIVE: To investigate the purification conditions of human anti-digoxin (AD) single chain antibody (ADAscFv) secreted and expressed in E.coli by combining FPLC with anion exchange chromatography. Methods: (1) The human ADAscFv was secreted into the culture medium by culture of E. coli HB2151, and the supernatant of the culture medium was prepared by centrifugation; (2) ammonium sulfate was added to collect the components whose initial saturation was 0 ~ 50% (3) The anion exchange column was connected with FPLC, loaded with Tris-HCl buffer containing NaCl and gradient elution gradient respectively, and the elution peak was detected by UV spectrophotometer. And SDS-PAGE were used to analyze the antigen binding activity and purity of human ADAscFv in the recovered samples respectively. Results: (1) There were 6 elution peaks in both elution methods. (2) ELISA showed the highest antigen-binding activity in the second elution peak. (3) SDS-PAGE showed that the second elution peak appeared to bind to human ADAscFv A single band of consistent molecular weight. Conclusion: This method can effectively purify human ADAscFv secreted by E. coli.