Specific Monoclonal Antibodies Recognizing the Endogenous Chicken High Mobility Group Box 1 Protein.

High mobility group box 1 (HMGB1) is a key member of the "danger associated molecular patterns" (DAMPs), which can localize in various compartments of the cell, and plays important roles in systemic inflammation. In the present study, monoclonal antibodies (MAbs) specifically against chicken HMGB1 were generated. The open reading frame of chicken HMGB1 was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-chHMGB1. The recombinant chicken HMGB1 protein was expressed in Escherichia coli Rosetta under IPTG induction and then purified by Ni-NTA Purification System. BALB/c mice were immunized with the purified recombinant HMGB1 protein, and three strains of hybridoma cells named 1F10, 8C11, and 4D8 secreting MAbs of chicken HMGB1 were obtained by hybridoma technique. Western blot and indirect immunofluorescence assays showed that the endogenous HMGB1 in various cell lines and glycosylated HMGB1 could both be specifically recognized by the prepared MAbs. This work indicated that the MAbs against chicken HMGB1 would be a valuable tool for further studies of HMGB1-mediated signaling in virus-infected cells and investigates the role of HMGB1 in avian virus pathogenesis.