Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha.

摘要: The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of diseases such as Crohn's disease, psoriasis, psoriatic arthritis, rheumatoid is basis for use anti-TNF-α therapies. neutralization TNF-α or blockage its binding to corresponding receptor has mainly served therapeutic strategy against some inflammatory diseases. This study aimed investigate production humanized single chain antibody (scFv) TNF-α. Therefore, murine monoclonal antibody, D2 mAb, was selected humanizing by complementarity determining region (CDR)-grafting method. Briefly, replacement CDRs from mAb with specific human scaffold led novel fragment variable (hD2). subsequent cloning hD2 into suitable expression vector, pGEX-6P-1, resulted 52-kDa GST-fusion protein E. coli, mostly form inclusion bodies. solubilization refolding GST-hD2 bodies achieved addition 4 M urea dialysis recover fusion soluble form. Then purified affinity chromatography through immobilized glutathione. GST pull-down experiment showed positive interaction between protein. Moreover, results an MTT assay that neutralizing activity (Kd 1.03 nM) hence potential be developed agent. However, more investigation needed elucidate in-vivo comparison other antibodies.