Yan Zhen, Lu Li, Shi Jihong, Bao Chunjie, Han Wei, Wu Yongjie, Zhang Yingqi
Biotechnology Center of Fourth Military Medical University, Chang Le Xi Lu #17, Xi'an 710032, Shaanxi, People's Republic of China.
Appl Biochem Biotechnol. 2006 May;133(2):149-62. doi: 10.1385/abab:133:2:149.
Interferon-alpha2a (IFN-alpha2a) has been used for the treatment of various viral infections and cancers for many years. However some untolerable side effects have limited its application in some aspects. To evaluate whether or not an oligopeptide containing GFE motif can home human IFN-alpha2a to specific tissues, a fusion gene was constructed by fusing the coding sequence of GFE peptide (CGFECVRQCPERC), which was screened from phage display peptide library, to the 3' end of human IFN-alphaa gene by recombinant DNA technique. Fusion protein rhIFN-alpha2a-GFE was expressed in Escherichia coli as inclusion bodies using a T7 RNA polymerase expression system, pET-22b, refolded through dialysis and purified to homogeneity to >95% of purity by affinity chromatography. Characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting demonstrated the authenticity of the fusion protein. Purified rhIFN-alpha2a-GFE was found to be functionally active in terms of its antiviral activity for about 2.5 x 108 IU/mg in vitro. Yields of the purified fusion protein were about 200 mg/L of culture medium. Tissue distribution assay in mouse showed that at 30 min IFN-alpha2a could be enriched sevenfold higher in lung in the targeted IFN group of mice than in the standard IFN group of mice, and last for a long time. At 1 h, IFN-alphaa in the targeted IFN group was still 4.02-fold higher than that in the standard group. This confirmed that GFE peptide has the ability to selectively deliver its fusion partner IFN-alpha2a to lungs. The results also showed that the IFN-alpha2a-GFE could be specifically enriched in kidney and liver. Its distribution in kidney was concordant with the finding of GFE receptor, MDP, in kidney. However, the IFN-alpha2a-GFE in liver may imply some significance in pharmacology and toxicology.
多年来，α2a干扰素（IFN-α2a）一直用于治疗各种病毒感染和癌症。然而，一些难以耐受的副作用在某些方面限制了其应用。为了评估含GFE基序的寡肽是否能将人IFN-α2a导向特定组织，通过重组DNA技术将从噬菌体展示肽库中筛选出的GFE肽（CGFECVRQCPERC）的编码序列融合到人IFN-α基因的3'端，构建了一个融合基因。使用T7 RNA聚合酶表达系统pET-22b在大肠杆菌中以包涵体形式表达融合蛋白rhIFN-α2a-GFE，通过透析复性，并通过亲和层析纯化至纯度>95%。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和免疫印迹进行表征，证明了融合蛋白的真实性。纯化的rhIFN-α2a-GFE在体外抗病毒活性方面具有功能活性，约为2.5×108 IU/mg。纯化融合蛋白的产量约为每升培养基200 mg。小鼠组织分布试验表明，在30分钟时，靶向IFN组小鼠肺中的IFN-α2a比标准IFN组小鼠富集高7倍，并持续很长时间。在1小时时，靶向IFN组中的IFN-α2a仍比标准组高4.02倍。这证实了GFE肽有能力将其融合伴侣IFN-α2a选择性地递送至肺。结果还表明，IFN-α2a-GFE可在肾脏和肝脏中特异性富集。其在肾脏中的分布与肾脏中GFE受体MDP的发现一致。然而，肝脏中的IFN-α2a-GFE可能在药理学和毒理学方面具有一定意义。
