Construction of adenoviral vector encoding soluble human TNFRI-IgGFc cDNA and its expression in human bronchial epithelial cells / 南方医科大学学报
Journal of Southern Medical University
; (12): 517-521, 2008.
Article
em Zh
| WPRIM
| ID: wpr-280159
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector carrying soluble extracellular region of tumor necrosis factor alpha receptor I-IgGFc (sTNFRI-IgGFc) and express the fusion protein in human bronchial epithelial HBE135-E6E7 cells.</p><p><b>METHODS</b>sTNFRI-IgGFc fusion gene was subcloned into the adenovirus shuttle plasmid pDC316, which was co-transfected with helper plasmid pBHGloxPE1,3Cre into HEK293 cells. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was generated by homologous recombination of the 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, and its titer measured using TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in the transfected HBE135-E6E7 were detected by RT-PCR and immunohistochemistry.</p><p><b>RESULTS</b>Ad-sTNFRI-IgGFc was successfully constructed with a viral titer of 3 x 10(10) TCID50/ml. The expression of sTNFRI-IgGFc mRNA and protein was confirmed in the transfected HBE135-E6E7 cells.</p><p><b>CONCLUSION</b>The constructed Ad-sTNFRI-IgGFc can effectively infect HBE135-E6E7 cells for efficient expression of sTNFRI-IgGFc protein, which antagonizes the cytolytic effect of TNFalpha in L929 cells, suggesting the potential of adenovirus expressing sTNFRI-IgGFc for local treatment of asthma.</p>
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1
Base de dados:
WPRIM
Assunto principal:
Proteínas Recombinantes de Fusão
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Brônquios
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Imunoglobulina G
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Fragmentos Fc das Imunoglobulinas
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Transfecção
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Adenoviridae
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Biologia Celular
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Receptores Tipo I de Fatores de Necrose Tumoral
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Células Epiteliais
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Vetores Genéticos
Tipo de estudo:
Prognostic_studies
Idioma:
Zh
Revista:
Journal of Southern Medical University
Ano de publicação:
2008
Tipo de documento:
Article