α干扰素-2bHis^58的分子构建、表达及纯化

Construction,Expression,and Purification of Interferon α-2bHis^(58)

目的通过定点突变,构建α干扰素-2bHis58(IFNα-2bHis58),以期获得高效的新型药物分子。方法采用PCR体外定点突变技术,使α干扰素-2b基因的第58位密码子由GAA突变为CAC。将扩增片段克隆入pET23b表达载体,重组质粒转化大肠杆菌BL21(DE3)。表达的IFNα-2bHis58经包涵体变复性、DEAE层析和CM层析纯化后,用WISH-VSV系统进行生物活性测定。结果IFNα-2bHis58主要以包涵体形式表达,表达量占菌体总蛋白的30%以上。纯化后,IFNα-2bHis58的纯度>95%,比活性>3.4×108IU/mg。结论构建了IFNα-2bHis58的表达载体,并成功在大肠杆菌中表达,获得了高活性突变分子IFNα-2bHis58,建立了IFNα-2bHis58的纯化工艺。

Objective To construct interferon α-2bHis^58 through site-directed mutagenesis in order to obtain a new type of molecule with higher potency. Methods GAA was substituted by CAC at codon 58 of interferon α-2b through PCR site-directed mutagenesis in vitro. The amplified fragments were inserted into pET23b expression vector,and the recombinant plasmid was transcribed into Eseherichia coli BL21 （DE3）. The bioactivity was determined by WISH-VSV system after the recombinant protein was purified through DEAE Sepharose Fast Flow and CM Sepharose Fast Flow. Results IFNα-2bHis^58 was expressed as inclusion bodies with the yield snore than 30% of total bacterial protein. The purity of IFNα-2bHis^58 was more than 95%,and the bioactivity was more than 3.4×10^8 IU/mg. Conclusion A interferon mutagenesis with higher efiqciency has been obtained. The construction, expression, and purification technology of IFNα-2bHis^58 has been established.