干扰素与转铁蛋白融合蛋白在毕赤酵母中的表达及鉴定  

Expression and Characterization of Fusion Protein of Interferon and Transferrin in Pichia pastoris

作　　者：李晓静[1] 张豪[1] 薛冲[1] 李彦英[1] 陈璟[1] 苗林[1] 方宏清[1] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物化学与生物物理进展》2005年第7期625-629,共5页Progress In Biochemistry and Biophysics

摘　　要：利用重叠PCR技术将干扰素(interferon,IFN)基因与转铁蛋白N端半分子(transferrinN-terminalhalf-molecule,TFN)基因在体外融合,融合基因和单独的TFN基因分别克隆至真核表达载体pPIC9中,转化毕赤酵母GS115,得到的转化子经诱导表达后在发酵上清中均获得了表达.经SPSepharoseFastFlow阳离子交换层析、PhenylSepharoseFastFlow疏水层析纯化,获得了纯度大于93%的重组融合蛋白IFN-TFN和纯度大于95%的重组TFN样品.生物活性实验证明融合蛋白IFN-TFN具有抗病毒活性.铁饱和实验证明融合蛋白IFN-TFN和单独的TFN具有相同的铁结合能力.因而TFN可望作为IFN的天然运输载体.The fused gene (IFN-TFN) of TFN (transferring N-terminal half-molecule) gene and IFN (interferon) gene was amplified by multiple PCR. The fused gene and TFN gene was inserted into pPIC9 vector. The recombinant plasmid pPIC9-IFN-TFN and pPIC9-TFN were transformed into Pichia pastoris GS 115 by PEG. After being induced by methanol, the target proteins were expressed in ferment supernatant at high level. The recombinant fused protein IFN-TFN and recombinant TFN with purity respectively being higher than 93% and 95% were finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. According to in vitro bioactivity assay, the fused protein IFN-TFN had antiviral activity but which was much lower than the natural IFN. Fe(3+) saturation study confirmed that the recombinant IFN-TFN was able to bind Fe(3+) as the recombinant TFN did. It was shown that TFN could be used as the transcellar carrier of IFN.

关 键 词：转铁蛋白 干扰素-Α 毕赤酵母 

分 类 号：R978.7[医药卫生—药品]