Gupta S, Eastman J, Silski C, Ferkol T, Davis P B
Department of Pediatrics, Case Western Reserve University School of Medicine, Biomedical Research Bldg, Rm 831, 2109, Adelbert Road, Cleveland, Ohio 44106-6006, USA.
Gene Ther. 2001 Apr;8(8):586-92. doi: 10.1038/sj.gt.3301451.
We have used an anti-human polymeric immunoglobulin receptor (pIgR) single chain Fv (scFv) to deliver reporter genes to epithelial cells in vitro. The scFv was constructed from a monoclonal antibody directed against pIgR and a cysteine residue was added at the carboxyl end to facilitate its conjugation to polylysine (polyK) via the heterobifunctional cross-linker SPDP. ScFv-cys was expressed in Drosophila S2 cells and purified to homogeneity using conventional column chromatography. ScFv-polyK, and polyK as control, were condensed with a DNA expression plasmid containing the luciferase reporter gene driven by the CMV promoter into unimolecular (with respect to DNA) complexes under high salt conditions. Target cells were MDCK cells transfected with human pIgR and repeatedly sorted for high-level receptor expression, with untransfected MDCK cells as control. Receptor-bearing MDCK cells were readily transfected by scFv-cys containing, pIgR directed complexes, and expression could be blocked by addition of excess human secretory component (SC), the extracellular portion of pIgR. In contrast, MDCK cells that did not express pIgR were not transfected. Nontargeted complexes were not effective in transfecting MDCK cells with or without pIgR. Targeted complexes also transfected human tracheal epithelial cells in primary culture, corroborating the pIgR-mediated gene delivery. These data indicate that a scFv directed against human pIgR can direct foreign genes specifically into receptor-bearing cells in vitro. We have expressed and purified a ligand that is efficient and specific in pIgR-mediated gene delivery.
我们已使用抗人多聚免疫球蛋白受体（pIgR）单链Fv（scFv）在体外将报告基因递送至上皮细胞。该scFv由针对pIgR的单克隆抗体制备而成，并在羧基末端添加了一个半胱氨酸残基，以便通过异双功能交联剂SPDP促进其与聚赖氨酸（polyK）偶联。ScFv-cys在果蝇S2细胞中表达，并使用常规柱色谱法纯化至均一性。在高盐条件下，将ScFv-polyK以及作为对照的polyK与含有由巨细胞病毒（CMV）启动子驱动的荧光素酶报告基因的DNA表达质粒浓缩成单分子（相对于DNA）复合物。靶细胞是转染了人pIgR并经反复分选以获得高水平受体表达的MDCK细胞，未转染的MDCK细胞作为对照。携带受体的MDCK细胞很容易被含有pIgR导向的scFv-cys复合物转染，并且加入过量的人分泌成分（SC，pIgR的细胞外部分）可阻断表达。相比之下，未表达pIgR的MDCK细胞未被转染。非靶向复合物对转染有无pIgR的MDCK细胞均无效。靶向复合物也能转染原代培养的人气管上皮细胞，证实了pIgR介导的基因递送。这些数据表明，针对人pIgR的scFv可在体外将外源基因特异性地导入携带受体的细胞。我们已经表达并纯化了一种在pIgR介导的基因递送中高效且特异的配体。
