Studies on interaction between hTNF-alpha and its two receptors with expressed hsTR55-preS1/hsTR75-preS1 fusion soluble receptors.
The gene encoding the N-terminal 2-50 amino acids of HBsAg-preS1 was amplified by PCR and fused to the 3'-end of two human soluble TNF receptor genes to form the hsTR55-preS1/hsTR75-preS1 fusion genes. The recombinant bicistronic expression vectors were further constructed, which contained one of the human soluble TNF receptor fusion genes and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), followed by the neomycin phosphotransferases as the selectable marker. BHK-21 cells transfected with those vectors by electroporation were selected with G-418, and the positive colonies expressing the protein of interest were obtained. All of the culture media of those transfects could fairly neutralize hTNFalpha-induced cytotoxicity to L929 cells. The expression of hsTR55-preS1/hsTR75-preS1 in those cells has been further demonstrated by RT-PCR and indirect ELISA at RNA transcription and protein translation levels. Their K(d) (dissociation constant) value has also been assayed with BIOSENSOR method. The results showed that the fused HBsAg-preS1 peptide did not affect the dissociation constant of hsTR55 or hsTR75 with hTNFalpha and its muteins. Thus, a novel nonradioactive ELISA method was developed for studies on interaction between hTNFalpha and its two receptors using those expressed fusion receptors.
Fang
Jia
J
Shanghai Research Center of Biotechnology, The Chinese Academy of Sciences, 200233, Shanghai, China. Jia_Fang@med.unc.edu
Lu
Fang
F
Wang
De Bao
DB
Chen
Chang Qing
CQ
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006801:Humans; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics; D017434:Protein Structure, Tertiary; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C050806:presurface protein 1, hepatitis B surface antigen