三种方法纯化生物工程抗体片段的比较

COMPARATIVE STUDIES ON PURIFICATION OF ENGINEERED ANTI-HBSFAB WITH 3 DIFFERENT METHODS

比较 3种不同纯化体系纯化生物工程抗 HBsFab的效率和效果 ,寻求高效、经济的生物工程产品批量纯化方法。采用增菌发酵抗 HBsFab阳性克隆 ,超声破菌法制备Fab上清。分别缓冲平衡抗Fab Sepharose亲合胶、链球菌蛋白G ( prot.G) Sepharose胶和Ni NTA离子螯合胶 ,对Fab上清进行过柱纯化。紫外光检测目的蛋白得率 ,SDS PAGE鉴定产物的纯度并用HBsAg包被ELISA检测其生物活性。结果显示 ,等容积不同类别的胶体对目的蛋白的纯化效率依次为 :Ni NTA离子螯合胶 >prot G Sepharose胶 >抗 Fab Sepharose亲合胶 ;在各胶体饱和结合能力之内 ,3种方法皆可获高纯度产品 ,在SDS PAGE中呈现单一区带 ;HBsAg包被ELISA检测结果为 3种方法纯化制备的Fab在抗原结合能力上未显示明显差别。提示 ,3种纯化方法各有优势和应用限制 ,Ni NTA法在经济性。

The purpose of this study was to compare the efficiency of 3 different methods in purifying engineered anti-HBsFab. Anti-HBsFab Supernatants containing anti-HBsFab were prepared by ultrasound lysis of host cells of anti-HBsFab positive clone. Anti-Fab-sepharose gel, streptococcal protein G-sepharose gel (Prot G gel) and Ni-NTA-agarose gel were used to purify Fabs according to the reagents protocols. Then the concentrations of purified proteins were determined. SDS-PAGE was used for the measurement of purified Fabs'purity. HBsAg based ELISA was chosen to determine the Fabs' bio-activity. The results indicated that Fab recovery of different gels in equal volume were different. The recovery of Ni-NTA GEL was greater than that of Prot G gel, and the recovery of Prot G gel was greater than that of anti-Fab gel. Purity of the Fab isolated by 3 different gels was confirmed by SDS-PAGE. The binding capacity of anti-HBs Fab to HBsAg of these three gels had no signifticant difference. Our data suggested that each method had its advantage and usage limitation, Ni-NTA method demonstrated much better performance in economy, management, and efficiency over the other two methods.