title: [Construction of adenoviral vector encoding soluble human TNFRI-IgGFc cDNA and its expression in human bronchial epithelial cells].
abstract: 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.
authors.affiliation: Department of Respiratory Diseases, Nanfang Hospital; School of Biotechnology, Southern Medical University, Guangzhou 510515, China. drsujin@126.com
authors.forename: Jin
authors.initials: J
authors.lastname: Su
authors.forename: Chang-xuan
authors.initials: CX
authors.lastname: You
authors.forename: Shao-xi
authors.initials: SX
authors.lastname: Cai
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Qian
authors.initials: Q
authors.lastname: Wen
authors.forename: Wei
authors.initials: W
authors.lastname: Luo
authors.forename: Yong-ta
authors.initials: YT
authors.lastname: Huang
mesh_terms: 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
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; C506235:TNFRSF1A protein, human