[Construction of adenoviral vector encoding soluble human TNFRI-IgGFc cDNA and its expression in human bronchial epithelial cells].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Su
Jin
J
Department of Respiratory Diseases, Nanfang Hospital; School of Biotechnology, Southern Medical University, Guangzhou 510515, China. drsujin@126.com
You
Chang-xuan
CX
Cai
Shao-xi
SX
Ma
Li
L
Wen
Qian
Q
Luo
Wei
W
Huang
Yong-ta
YT
D000256:Adenoviridae / Q000235:genetics*; D001980:Bronchi / Q000166:cytology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; C506235:TNFRSF1A protein, human