single chain Fv;
plgR;
gene delivery;
epithelial cells;
D O I:
10.1038/sj.gt.3301451
中图分类号:
Q5 [生物化学];
Q7 [分子生物学];
学科分类号:
071010 ;
081704 ;
摘要:
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.
机构:
Seoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South KoreaSeoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South Korea
Park, In Young
Kim, In Yong
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Seoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South KoreaSeoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South Korea
Kim, In Yong
Yoo, Mi Kyong
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Seoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South KoreaSeoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South Korea
Yoo, Mi Kyong
Choi, Yun Jaie
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Seoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South KoreaSeoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South Korea
Choi, Yun Jaie
Cho, Myung-Haing
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Seoul Natl Univ, Coll Vet Med, Seoul 151742, South KoreaSeoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South Korea
Cho, Myung-Haing
Cho, Chong Su
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Seoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South KoreaSeoul Natl Univ, Res Inst Agr & Life Sci, Sch Agr Biotechnol, Seoul 151921, South Korea