Production and purification of recombinant HBcAg and investigation of its structural and functional characteristics

Hepatitis B, as a major healthcare problem and a potentially life-threatening infection, is caused by Hepatitis B virus (HBV). Nucleocapsid of HBV is composed of a single polypeptide chain known as core antigen, HBcAg. As a highly immunogenic subviral particle, HBcAg has been used as a carrier platform for heterologous proteins. Anti-HBc antibody, as one of the serological markers for HBV infection, is the best marker for documenting prior exposure to HBV. Here, we produced, purified and characterized HBcAg. pColdI vector, containing a histidine tag, was used for the first time to clone HBcAg gene. The highest level of HBcAg expression was achieved in Tuner strain, followed by BL21 and Rosetta-gami. Histidine-tagged HBcAg was purified using Ni-NTA resin, and its structural and antigenic similarity with natural HBcAg was confirmed by ELISA and western blot. Then, recombinant HBcAg was used to produce anti-HBc polyclonal antibodies in rabbits, and specific anti-HBcAg polyclonal antibodies were purified by the HBcAg-CNBr sepharose column. Specific anti-HBcAg polyclonal antibodies were able to detect recombinant HBcAg up to 0.15 ng/ml in an optimized sandwich ELISA. The results of this study provide valuable information for the production of recombinant HBcAg and specific anti-HBcAg polyclonal antibodies for further research and diagnostic applications.