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Journal of Southern Medical University ; (12): 517-521, 2008.
Article Dans Chinois | WPRIM | ID: wpr-280159

Résumé

<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>


Sujets)
Adenoviridae , Génétique , Bronches , Biologie cellulaire , Cellules épithéliales , Biologie cellulaire , Métabolisme , Vecteurs génétiques , Génétique , Fragments Fc des immunoglobulines , Génétique , Immunoglobuline G , Génétique , Récepteur au facteur de nécrose tumorale de type I , Génétique , Protéines de fusion recombinantes , Génétique , Transfection
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