Studies on interaction between hTNF-α and its two receptors with expressed hsTR55-preS1/hsTR75-preS1 fusion soluble receptors

Abstract

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 hs TR55- preS1/hs TR75- 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 hTNFα-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 hTNFα and its muteins. Thus, a novel nonradioactive ELISA method was developed for studies on interaction between hTNFα and its two receptors using those expressed fusion receptors.