固定化木瓜蛋白酶水解HCG抗体及其片段的纯化

Hydrolysis of HCG Monoclonal Antibodies with Immobilized Papain and Purifi cation of Its Fragments

采用温敏载体NHS-PNIPAM固定化木瓜蛋白酶,水解β-HCG单克隆抗体制备Fab片段,并优化水解工艺。结果表明:30℃,在pH 7.0的磷酸盐缓冲液中(含10 mmol/L半胱氨酸,2 mmol/L EDTA),加酶量40 u·(mg抗体)-1,转速180 r/min,反应3 h,β-HCG抗体的转化率大于98.0%,主要生成目的产物Fab片段。酶解产物通过Protein L亲和色谱法分离纯化,得到的Fab片段回收率为74.5%,纯度为98.6%。

HCG monoclonal antibody was hydrolyzed by papain immobilized in NHS-PNIPAM and the hydrolysis process was optimized to prepare Fab fragments. The results showed that 1 mg HCG antibodies were hydrolyzed to Fab fragments with conversion rate over 98.0 % by 40 u immobilized papain active for 3 h at 30℃, pH 7.0, 180 r/min in 1 ml phosphate buffer solution （pH 7.0） containing 10 mmol/L cysteine and 2 mmol/L EDTA. Fab fragments were purified by Protein L affinity chromatography from the hydrolysis mixture. The purity of the obtained Fab fragments was 98.6 % with the recovery of 74.5 %.