Soluble Tumor Necrosis Factor Receptor Stnfr55 Gene Cloning And Its Antagonist Screening Methods Research

Tumor necrosis factor(TNF) is a potent cytokine that elicits a broad spectrum of biological responses. These responses are mediated by two distinct receptors on cell surface with molecular weight of 55KDa and 75KDa. Soluble TNF receptors can be shed proteolyticly from the two receptors on cell surface. In this study, soluble TNF receptor sTNFR55 was expressed secretively in insect cell line and Streptomyces lividans separatedly. A model for screening TNF receptor antagonists from natural products has been developed with the recombinant sTNFR55 as target.Total RNA was extracted from Hela cells by Acid Guanidine Isothiocyanate-Phenol-Chloroform. The cDNA of sTNFR55 was amplified from the total RNA by RT-PCR technique. The cDNA was cloned into baculovirus vector of insect cell and plasmid pIJ459 of S. lividans respectively.1. Insect cell system: The 530bp cDNA was cloned into transfer plasmid pAcGP67B. and subcloned into pUC19 for DNA sequencing. Sequencing result of cDNA showed that there are two base differences at position 64 and 173 between the amplified cDNA and sTNFR55 sequence reported before. The recombinant plsmid pAcTNFR(pAcGP67B-sTNFR55) and wild type AcMNPV DNA were cotransfected into insect cell line sf9 through a modified calcium phosphate precipitation method. Recombinant baculovirus AcMNPV-TNFR was formed after homologous recombination between baculovirus and pAcTNFR in vivo. The purified AcMNPV-TNFR was obtained with end-point dilution-Dot blot and plaque assay. Dot blot and PCR confirmed that AcMNPV-TNFR contained the sTNFR55 gene.SDS-PAGE, receptor-ligand blot assay, neutralization of TNF...