Construction of adenoviral vector encoding soluble human TNFRI-IgGFc cDNA and its expression in human bronchial epithelial cells.
- Author:
Jin SU
1
,
2
;
Chang-xuan YOU
;
Shao-xi CAI
;
Li MA
;
Qian WEN
;
Wei LUO
;
Yong-ta HUANG
Author Information
- Publication Type:Journal Article
- MeSH: Adenoviridae; genetics; Bronchi; cytology; Epithelial Cells; cytology; metabolism; Genetic Vectors; genetics; Immunoglobulin Fc Fragments; biosynthesis; genetics; Immunoglobulin G; biosynthesis; genetics; Receptors, Tumor Necrosis Factor, Type I; biosynthesis; genetics; Recombinant Fusion Proteins; biosynthesis; genetics; Transfection
- From: Journal of Southern Medical University 2008;28(4):517-521
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSsTNFRI-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.
RESULTSAd-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.
CONCLUSIONThe 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.
