一种重组人α1型干扰素突变体IFN-α1/86D的纯化与鉴定  

PURIFICATION AND CHARACTERIZATION OF A NOVEL ENGINEERED MUTANT OF HUMAN α1-TYPE INTERFERON--IFN-α1/86D

作　　者：周圆[1] 金冬雁[1] 张智清[1] 王伟[1] 黎志良[1] 迟捷 李玉英[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室,北京100052

出　　处：《病毒学报》1993年第1期37-42,共6页Chinese Journal of Virology

基　　金：国家863高科技生物技术领域资助

摘　　要：本文研究了重组人α1型干扰素突变体IFN-α1/86D在摇瓶培养和分批发酵培养的重组大肠杆菌DH5α株中的表达动态,并采用阳离子交换层析和抗α1型干扰素单克隆抗体亲和层析的两步流程对其进行了纯化,得到SDS-PAGE纯及高效液相层析(HPLC)纯的IFN-α1/86D产品,共比活性为2.3×10~7单位/毫克蛋白。N端氨基酸序列测定的结果表明,纯化产品的纯度在95％以上,但其N端不均一,产品中含有两种N端序列正确的IFN-α1/86D活性分子,即约75％为去Met分子和约25％为带Met分子。The kinetics of the expression of a novel engineered mutant of hu-man α1-type interferon ( IFN-α1/86D)in E.coli grown in shaken flasks or fermentor was studied. The target protein was then purified to>95% homogeneity through cation exchange chromatograpy ( CM-Sepharose ) and affinity chromatography ( monoclonal antibody against IFN-α1 ) , the purified protein with a specific activity of 2.3×107 IU/mg protein, appeared as a single band on the SDS-PAGE electrophoretogram and a single peak on the HPLC chromatogram. The N-terminal amino acid se-quence of purified recombinant human IFN-α1/86D was determined on Applied Biosystems 477A protein/peptide sequencer and was shown to be correct. The purified protein was demonstrated to be heterogenous, co mprising of two sequences which differ merely by the N-terminal Met residue-25% Met-plusspecies and 75% Met-minus.

关 键 词：干扰素 蛋白 提纯 抗病毒活性 

分 类 号：R978.7[医药卫生—药品]