title: [Expression of human tumor necrosis factor receptor-Fc fusion protein and characterization of its biological activity].
abstract: OBJECTIVE
  To construct a recombinant adeno-associated virus vector carrying the fusion product of extracellular domain of human tumor necrosis factor receptor (hTNFR) with human IgG Fc fragment, and detect the expression and biological activity of hTNFR-Fc.
  
  
  METHODS
  The recombinant hTNFR-Fc fusion gene was cloned into adeno-associated virus (AAV) vector, which was used to transfect human embryo kidney cells (HEK293T) in vitro. The expression of the target protein hTNFR-Fc in the supernatants of the transfected HEK293T cells was identified by Western blot analysis. TNF-α inhibition assay in vitro was performed using TNF-α-sensitive mouse fibrosarcoma L929 cells to determine the neutralizing capacity of hTNFR-Fc against human or rat TNF-α. The binding activity of hTNFR-Fc to human or rat TNF-α was identified by ELISA.
  
  
  RESULTS
  The recombinant plasmid pAAV/hTNFR-Fc was constructed and the target protein of hTNFR-Fc was expressed in the supernatants of the transfected HEK293T cells. It could effectively bind to human TNF-α and neutralize its cytotoxicity to L929 cells. The hTNFR-Fc protein also exerted the binding and neutralization activities to rat TNF-α to some extent.
  
  
  CONCLUSIONS
  The study constructed hTNFR-Fc fusion gene and verified its secretory expression in AAV vector, as well as identified its biological activities, which lay the foundations for future viral package and animal experiments.
authors.affiliation: Department of Translational Medicine, Beijing Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Zhiyan
authors.initials: Z
authors.lastname: Du
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.forename: Jingying
authors.initials: J
authors.lastname: Zhu
authors.forename: Jiyun
authors.initials: J
authors.lastname: Yu
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D051381:Rats; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
chemical_list: D007141:Immunoglobulin Fc Fragments; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha