抗人肝癌单抗HAb18F(ab’)_2片段的制备及纯化

Preparation and Purification of F(ab')_2 Fragment of anti hepatoma McAb HAb18

目的：制备抗人肝癌单抗（ ＭｃＡｂ） ＨＡｂ１８ Ｆ（ａｂ’）２ 片段。方法 ：用二硫苏糖醇（ＤＴＴ） 激活木瓜蛋白酶，ｐＨ５ ．５ 环境下消化ＨＡｂ１８ ＭｃＡｂ（ＩｇＧ１） ，然后用快速蛋白液相色谱（ＦＰＬＣ） ＤＥＡＥ－ Ｓｅｐｈａｒｏｓｅ － ＦＦ 柱（２ｃｍ ×１８ｃｍ）纯化。对所获Ｆ（ａｂ’）２ 段的纯度、免疫活性、热原质、细菌、产率等方面进行质量检测。结果：在酶∶抗体为１∶２０ ，消化ＨＡｂ１８ ２ ｈ ，９５ ％ 以上ＩｇＧ 裂解为Ｆ（ａｂ’）２ 段，纯化后Ｆ（ａｂ’）２ 段的纯度为＞ ９６ ．１ ％ ，纯化周期６０ ｍｉｎ ， 一次制备量可达５００ ｍｇ ，产率为５３ ％ ，免疫活性１∶８０００ ，细菌、热原检测结果为阴性。结论：该法是一种快速、大量制备高产率、高质量ＭｃＡｂ ＩｇＧ１ 亚类Ｆ（ａｂ’）２ 段的有效方法。

Objective:The aim is to prepare F(ab) 2 fragment of anti hepatoma McAb HAb18. Methods: Papain was preactivated with DTT and then separated from excess DTT. HAb18 McAb IgG1(10 mg/ml) was incubated with preactivated papain(2 mg/ml)at 37℃ in 3mM EDTA,0.1 M acetate pH5.5. The papain digests were dialyzed against 10 mmol/L Tris HCl, pH8.0 and applied to a column of DEAE sepharose FF(2 cm×18 cm) FPLC which is equilibrated with the same buffer, and eluted with a linear gradient of NaCl from 0 to 100 mmol.Results: More than 95% McAb HAb18 IgG1 was cleavaged after incubation for 2h at the papain to IgG ratio of 1∶20(w/w). The purity of F(ab) 2 fragment was about 96.1% by sodium dodycel sulfate polyacrylamide gel electrophoresis(SDS PAGE).The recoveryof the antigen binding site was 53%,and immunoactivity was 1∶8000 by inteact immunoflurescence.The cycle time of the DEAE Sepharose FF FPLC was 60 min, and up to 500 mg F(ab) 2 fragment was purified in a cycle.Conclusion:This method was suitable especially for large scale preparation and purification of McAb IgG1 F(ab) 2 fragment. Key words antibody, monoclonal Papain Chromatography,ion exchange Liver neoplasms YU Rang\|cai\ CHEN Qiao\|ling\ LIU Wei (College of Biotechnology,South China Agricultural.University,Guangdong Prov.Guangzhou,510642) ABSTRACT:The straw mushroom( Volvariella volvacea) were irradiated by 60 Co γ ray of different doses and stored at 16℃.The experimental results showed that the irradiation treatment inhibited membrane lipid peroxidation and decreased the membrane leakage,therefore,improve the fresh keeping effect of straw mushroom.