Preliminary Exploration, Blocking Il-1 And Tnf-¦Á Treatment Of Rheumatoid Arthritis

In this study, we aimed to find a way to inhibit the binding of IL-1 and TNF-a to their receptors on the cytomembrane which was supposed to be a critical cause in RA.IL-1ra was amplified using error-prone PCR.The amplified products were cloned into pHB-1SVFV to construct IL-lra mutant library. Phage displaying technique was used to select some IL-lra mutants which can bind IL-1 receptor more effectively. After phage displaying and panning, 11 mutants were selected from the library.Then the mutants were cloned into plasmid pTIG-Trx. The recombinant plasmids were expressed in E.coli BL21(DE3) with the induction of IPTG, we have obtained 10 mutant proteins. Some of them were expressed solubly but others were in the form of inclusion bodies. Western-blot analysis showed that the mutants retained their binding capacity to IL-1ra antibodies. All of the mutants were purified by Ni2+ chelate chromatography and gel filtration chromatography. We defected the bioactivity of IL-1ra and its mutants using EL-4 and CTLL-2 cells. Results showed that the affinity of 40-22, 40-4 and 50-4 were higher than wtlL-1ra.In order to block TNF-a binding to its receptor we constructed the soluble tumor necrosis factor receptor II 15-157-IgG hinge region chimeric protein. First we extracted the total RNA of U937 cells, then we amplified TNFRII 15-157 cDNA by RT-PCR, and constructed TNFRII - 15-157:Fc-hinge region (F:R II 15-157-FcH) by overlapping PCR.The recombined sequence was inserted into pTIG-Trx vector, and expressed in E.coli BL21(DE3) with the induction of IPTG The products were soluble and functional. The bioactivity of F:RII 15-157-FcH was tested by MTT cell survival assay: F:RII 15-157-FcH can block the cytotoxity of TNF on mouse L929 cells.Finally we established the collagen-induced arthritis to determin the bioactivity of IL-lra, IL-lra 40-22 and F:RII 15-117-FcH in vivo. The result showed that all of them exhibited the ability to lighten the symptom of RA, and a better effect was given when IL-lra 40-22 and F:RII 15-157-FcH were given together.The studying of IL-lra, IL-lra mutants and F:RII 15-157-FcH provides clues forfurther research of the treatment of RA.