Hospital & Institute of Hepatobiliary Surgery, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853, China; Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Cell Biochem Funct. 2013 Dec;31(8):698-706. doi: 10.1002/cbf.2958. Epub 2013 Mar 19.
Liver-directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi-functional targeting molecule, which maintains targeting, endosome-escaping, and DNA-binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full-length cDNA encoding anti-hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50-Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC-C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv-Mel-Gal4 triple fusion protein (C1MG) was purified with a Ni(2+) chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni(2+) column purification. Western blot and immunohistochemistry showed the antigen-binding ability of C1MG to the cell surface of the liver-derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane-disrupting activity. DNA-binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome-escaping and DNA-binding capacity, which makes it probable to further study its liver-specific DNA delivery efficacy in vivo.
肝靶向基因治疗已成为许多肝脏疾病的一种有前途的治疗方法。在本研究中，我们构建了一种多功能靶向分子，该分子保持了靶向、内体逃逸和 DNA 结合能力，用于基因传递。合成了两种蜂毒素（M）的单链寡核苷酸。从 C1/pIT2 中纯化了编码抗肝去唾液酸糖蛋白受体 scFv C1（C1）的全长 cDNA。通过聚合酶链反应从 pSW50-Gal4 扩增 GAL4（G）基因。将 M、C1 和 G 插入质粒 pGC4C26H 中，产生重组质粒 pGC-C1MG。融合基因 C1MG 随后亚克隆到质粒 pET32c 中，产生重组质粒 C1MG/pET32c，并在大肠杆菌 BL21 中表达。用 Ni（2+）螯合 HiTrap HP 柱纯化 scFv-Mel-Gal4 三融合蛋白（C1MG）。融合蛋白 C1MG 约 64kD 以包涵体形式表达；用 Ni（2+）柱纯化制备 4.5mg/ml C1MG。Western blot 和免疫组化显示 C1MG 与肝源性细胞系和肝组织切片表面的抗原结合能力。溶血试验表明 C1MG 保持了膜破坏活性。通过荧光素酶测定证实了 C1MG 的 DNA 结合能力，表明 C1MG 可以基于 C1MG 有效地将 DNA 递送到细胞中。在大肠杆菌中成功表达了 C1MG，并且 C1MG 重组蛋白赋予了靶向、内体逃逸和 DNA 结合能力，这使得进一步研究其在体内的肝特异性 DNA 递送效果成为可能。
