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
in Chinese
| WPRIM
| ID: wpr-280159
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>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Fusion Proteins
/
Bronchi
/
Immunoglobulin G
/
Immunoglobulin Fc Fragments
/
Transfection
/
Adenoviridae
/
Cell Biology
/
Receptors, Tumor Necrosis Factor, Type I
/
Epithelial Cells
/
Genetic Vectors
Type of study:
Prognostic study
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2008
Type:
Article
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