Tian Shuo, Li Qinshan, Yao Wenbin, Xu Chen
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China.
Protein Expr Purif. 2013 Aug;90(2):124-8. doi: 10.1016/j.pep.2013.05.002. Epub 2013 Jun 6.
The main obstacles to using Interferon a1 as an antiviral agent are its low stability and fast clearance. Here, we prepared a long-lasting recombinant human serum albumin-interferon α1 fusion protein. It was expressed in methylotrophic yeast Pichia pastoris with HSA's natural signal peptide and purified by dye affinity chromatography and ion exchange chromatography. The physicochemical, biological, and pharmaceutical characteristics of HSA-IFN α1 were then evaluated in vitro and in vivo. The purity of HSA-IFN α1 was about 95% analyzed by SDS-PAGE. Molecular weight determined by MALDI-TOF mass spectrometry was 86,582. Western blot analysis showed that the expressed HSA-IFN α1 had the antigenicity of HSA. The N-terminal acid amino sequence was identical to predicted sequence. The antivirus activity in vitro evaluated by cytopathic effect assay was (1.63±0.06)×10(5) IU/mg. After administered in rats, the biological activity of HSA-IFN α1 were enhanced and maintained for 6days in serum. Overall, these studies demonstrate the feasibility of using albumin-fusion technology to development a long-lasting, antiviral protein HSA-IFN α1 with high antivirus activity.
将干扰素α1用作抗病毒药物的主要障碍是其稳定性低和清除速度快。在此，我们制备了一种长效重组人血清白蛋白-干扰素α1融合蛋白。它在甲基营养型酵母毕赤酵母中利用人血清白蛋白的天然信号肽进行表达，并通过染料亲和色谱和离子交换色谱进行纯化。然后在体外和体内评估了人血清白蛋白-干扰素α1的物理化学、生物学和药学特性。通过SDS-PAGE分析，人血清白蛋白-干扰素α1的纯度约为95%。通过基质辅助激光解吸电离飞行时间质谱测定的分子量为86,582。蛋白质印迹分析表明，表达的人血清白蛋白-干扰素α1具有人血清白蛋白的抗原性。N端氨基酸序列与预测序列相同。通过细胞病变效应试验评估的体外抗病毒活性为(1.63±0.06)×10(5) IU/mg。在大鼠体内给药后，人血清白蛋白-干扰素α1的生物学活性增强并在血清中维持6天。总体而言，这些研究证明了使用白蛋白融合技术开发具有高抗病毒活性的长效抗病毒蛋白人血清白蛋白-干扰素α1的可行性。
