Expression and maturation of Sendai virus vector-derived CFTR protein: functional and biochemical evidence using a GFP-CFTR fusion protein
| Autor: | Makoto Inoue, H Ban, Akihiro Iida, Eric W.F.W. Alton, Mario Chan, Mamoru Hasegawa, Felix M. Munkonge, Uta Griesenbach |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Cystic Fibrosis
Recombinant Fusion Proteins viruses Genetic Vectors Green Fluorescent Proteins Cystic Fibrosis Transmembrane Conductance Regulator Gene Expression Respiratory Mucosa Sendai virus Cell Line law.invention Green fluorescent protein Mice Transduction (genetics) Transduction Genetic law Gene expression Genetics Animals Molecular Biology Mice Inbred BALB C Microscopy Confocal biology Genetic Therapy respiratory system biology.organism_classification Fusion protein Virology Transmembrane protein Cystic fibrosis transmembrane conductance regulator Cell biology Perfusion Recombinant DNA biology.protein Molecular Medicine Nasal Cavity |
| Zdroj: | Gene Therapy. 14:1688-1694 |
| ISSN: | 1476-5462 0969-7128 |
| Popis: | Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|