Affiliations:
Department of Medical Biotechnology, Biotechnology Center of Ho Chi Minh City, Trung My Tay Ward, HCM City, Vietnam
Correspondence:
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Corresponding author: Quan Dang Nguyen, Department of Medical Biotechnology, Biotechnology Center of Ho Chi Minh City, 2374 Highway 1, Trung My Tay Ward, District 12, HCM City, Vietnam. E-mail: [email protected].
Abstract: BACKGROUND:TNF-α (Tumor necrosis factor-alpha) plays a vital role in the human immune system. However, abnormal activity and overexpression of TNF-α are some of the causes of allergic and autoimmune diseases. Inhibiting the activity of this molecule is one of the novel pathologies for these diseases. The development of this recombinant protein is expected to reduce the financial burden of treating inflammatory rheumatic diseases. OBJECTIVE:The study’s objective was to generate and evaluate the biological activity of TNFR-Fc, construct of fusions an extracellular part TNF-α receptor (p75) and an Fc fragment of human immunoglobulin G1, expressed from the CHO-DG44 cell system. METHODS:The recombinant TNFR- Fc plasmid was constructed and identified by PCR, restriction enzyme digestion, and sequencing. A stable cell line for expression of TNFR-Fc was selected by limiting dilution cloning. Structural characterization, the binding affinity of TNFR-Fc to TNF-α, the neutralizing the cytotoxic activity- induced TNF-α, and the TNF-α-induced apoptosis suppression of TNFR- Fc were analyzed by SDS/PAGE Western blotting, ELISA, WST assay, Immunofluorescence, and flow cytometry. RESULTS:Preliminary analysis of the structural characteristics showed that TNFR-Fc is a low- glycosylated protein and perhaps in dimeric form. Furthermore, the recombinant TNFR-Fc can interact with its ligand TNF-α with a dissociation constant Kd 0.25±0.03μM equivalent to that of the original drug, Etanercept. We also demonstrated that TNFR-Fc expressed from CHO-DG44 was able to neutralize TNF-α- induced cytotoxic activity and inhibited p53-related apoptosis in vitro, similar to Etanercept. CONCLUSIONS:These data collectively suggested that TNFR-Fc potently blocks TNF-α, which could be a novel therapeutic strategy for cytokine-driven diseases.
