Purification and identification of recombinant human interferon-lamda 1 / 中国组织工程研究

ABSTRACT

BACKGROUND:In 2003,a new family of interferon(IFN) III is discovered,that is,IFN-?s including three subtypesIFN-?1,2 and 3.OBJECTIVE:To analyze the recombinant human interferon-?1(rhIFN-?1) expressed in Pichia pastoris,further more,to identify its physical and chemical characteristics,expression of receptors,and anti-tumor activities.DESIGN,TIME AND SETTING:The single sample observation was performed at the Department of Biochemistry and Molecular Biology,Institute of Basic Medical Sciences from September 2006 to July 2008.MATERIALSThe barms pAO815-4?F-IFN?1/GS115 contained rhIFN?1 expressive plasmids,human cervical carcinoma cell Hela and HCT116 cells were preserved in the state key lab for Molecular Biology from Institute of Basic Medical Sciences Chinese Academy of Medical Sciences METHODS:The Pichia pastoris contained rhIFN-?1 expressive plasmids was cultured and induced to express IFN-?1.The positive ion exchange chromatography and gel chromatography were used to purify the expression product.MAIN OUTCOME MEASURES:The physico-chemical characters of the purified product were tested.The product's IL-10R2 and IL-28R were investigated by RT-PCR and real-time quantitative PCR and its antitumor activity was tested by MTT analysis.RESULTS:The expressed product rhIFN-?1 yield reached to 9.23 mg/L,and its purity was near 90%.The isoelectric point of rhIFN-1 was 8.01,with molecular weight of 20 960.The expressions of IL-10R2 and IL-28R were detected in U937 cell.rhIFN-?1 could decrease the cell proliferation just like IFN-?2a,but this function was unclearly in the IL-28R cell.CONCLUSION:rhIFN-?1 can be highly expressed in Pichia pastoris,and its product has the physical and chemical characteristics consistent with natural IFN-?1,and the anti-tumor activity is similar to IFN-?2a,however,it is weak in decreasing the proliferation of relatively lowerintensity of receptor distribution in marrow-hematopoiesis system.