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In Silico and In Vitro Investigations On Mutated IFNβ-scFv Against Human Collagen Type II


 Current medications for rheumatoid arthritis (RA), a common synovial autoimmune disease, are associated with adverse effects. Interestingly, interferon beta (IFNβ), effective in multiple sclerosis (MS) treatment, also can help decreasing articular destruction in RA. Here, a novel fusion protein was introduced containing human mutated IFNβ (with mutations in 27th and 101th residues; IFNβ27+101) fused to a single chain fragment variable (scFv) antibody against human collagen type II for decreasing IFNβ27+101 off-targets (according to drug targeting benefits) in future in vivo and clinical experiments. After designing, bioinformatic analyses and the recombinant vector transfection into HEK293 cells, the mutated IFNβ-scFv protein confirmation and function were assessed by SDS-PAGE, western blotting, ELISA, and real-time PCR. The fusion protein secondary and tertiary structures had proper folding. Also, the recombinant mRNA secondary structure considered stable. 2.35 fold difference between the test and negative control groups confirmed the scFv attachment to human collagen type II (p= 0.046). MxA 25.68 fold overexpression in peripheral blood mononuclear cells (PBMCs) treated with the recombinant protein compared with the non-treated sample (p= 0.0001), demonstrated IFNβ27+101 bioactivity as the fusion protein. In vitro and in silico studies verified function of mutated IFNβ-scFv, however in vivo studies are proposed for further validation.