Cloning, Expression And Bioactivity Detecting Of The Extracellular Region Of Human IL-1RI CDNA

IL-1 is a critical pro-inflammatory cytokine which is concerned with Rheumatoid arthritis (RA).The aim of the present study is to find a way to inhibit the binding of IL-1 to its receptors on the cytomembrane which was supposed to be a critical cause in RA.The total RNA was extracted from HepG2 cell. The extracelluar cDNA (957bp) of human type I interleukin-1 receptor (IL-1RI) was amplified by RT-PCR and nest PCR with total RNA of HepG2 as the template. After comparing the sequence data of sIL-1RI gene with that of Gene Bank, the sequence data obtained from our experiment is consistent with that of Gene Bank. The extracelluar cDNA of human IL-1RI was ligated into pTIG-Trx vector and pPICZaA vector respectively, which were expressed in prokaryotic and eukaryotic expression systems.In prokaryotic expression system, the extracellular cDNA of human IL-1RI was ligated into vector pTiG-Trx. The protein of sIL-1RI was expressed by the induction of IPTG in E.coil BL21 (DE3), which was transferred with plasmid of pTIG-Trx/sIL-1RI. The conditions for the expression of the target protein were optimized, when the culture reached an OD₆₀₀=1.0 it was induced with 1.0mmol/L IPTG at 20℃ for almost 20 hours. The results of SDS-PAGE demonstrated that some of them were soluble but others were in the form of inclusion bodies. The target protein was detected by Western Blot and ELISA analysis. To obtain the active target protein, the inclusion bodies were dissolved with SKL and renaturated by ladder dialysis. And then we concentrated inclusion badies by ultrafiltration.The bioactivity of the protein of sIL-lRI were detected by MTT cell survival assay using EL-4 and CTLL-2 cells. Results showed that the target protein had the binding capacity to IL-1.In eukaryotic expression system, the target gene was cloned into pPICZaA vector. After the plasmid sIL-1RI/pPICZαA has been linearized by restriction enzyme SacI, we used electroporation for transformation of Pichia pastoris GS115. Add 100% methanol to a final concentration of 0.5% methanol every 12 hours to maintain induction after the culture reached an OD₆₀₀=1.0. All expression was done at 30℃ for 60-72 hours in a shaking incubator. For secreted expression, we analyzed the supernatants by SDS-PAGE and the results of SDS-PAGE showed a prominent band at 43kDa. The target protein was detected by ELISA analysis. The bioactivity of the protein of sIL-1RI was determined by MTT cell survival assay using EL-4 and CTLL-2 cells.Results showed that the target protein had the banding capacity to IL-1.