干扰素与转铁蛋白融合蛋白在毕赤酵母中的表达及鉴定

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 (transferrin N-terminal half-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 SP Sepharose Fast Flow 阳离子交换层析、 Phenyl Sepharose Fast Flow 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 .

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

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 GS115 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. Fe3+ saturation study confirmed that the recombinant IFN-TFN was able to bind Fe3+ as the recombinant TFN did. It was shown that TFN could be used as the transcellar carrier of IFN.