人rIFN-γ的纯化及鉴定实验研究

PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN r-INTERFERON-γ PRODUCED IN ESCHERICHIA COLI

本研究利用本室建株的A_3 杂交瘤细胞株分泌的单克隆抗体,对大肠菌表达的人rIFN-γ进行了纯化。宿主菌经 7MGuHCl 处理后,收获的抽提液经 40％饱和(NH_4)_2SO_4盐析,除去部分杂蛋白,纯度约提高了一倍、活性回收>100％。此溶液稀释70倍复性后直接通过McAb亲和层析纯化,纯度达到98％,比活6×10~6Iu/mgp。本文还对复性的影响因素进行了研究,证实溶液的离子强度及蛋白浓度对复性均有影响。

A method applying McAb from A hybrid tumor cells for purification of human r-IFN-γ derived from E. colicells was developed in our laboratory. The E. coli cells producing r-IFN-γ were treated wilh 7M GuHCl and the extract was salted with 40% (NH4)2SO4 partially to eliminate the contaminating protein, which increased the purity of the extract from 23% to 41% with 100% activity recovery. This partially purified extract was diluted 70 fold and purified by McAb affinity chromatography with a specific activity of 6 × 106IU/mgp. Every 5ml McAb-Sephadex 4B prepared in this study could absorb a minimum of 1.5mg r-IFN-γ. The ionic strength of the diluent for the extract was an important factor in the renaturation, 0.02M PBS being the most appropriate.Reduced SDS-PAGE silver-staining and western blotting analyses showed a major band at MW 17,000 and a Very minor band at MW 34,000; both representing r-IFN-γ.