Prokaryotic expression of recombinant human bone morphogenetic protein-2 and preparation of monoclonal antibodies / 中国医学科学院学报

ABSTRACT

<p><b>OBJECTIVE</b>To express and purify the recombinant human bone morphogenetic protein-2 mature peptide (rhBMP-2m) in prokaryotic system and to develop highly-specific monoclonal antibodies.</p><p><b>METHODS</b>An engineered E. coli strain expressing rhBMP-2m was fermented. The bacterial cells were firstly lysed and then the rhBMP-2m inclusion bodies were isolated by centrifugation. After the inclusion bodies had been solubilized by high-concentration denaturing agents, denatured rhBMP-2m was purified by cation ion-exchange chromatography. Biologically active rhBMP-2m was obtained by refolding of purified denatured rhBMP-2m through direct dilution. The refolded rhBMP-2m was used to immunize Balb/c mice to develop anti-rhBMP-2m monoclonal antibodies using classic hybridoma technique.</p><p><b>RESULTS</b>rhBMP-2m with a purity greater than 95% was obtained on reduced SDS-PAGE. The refolded rhBMP-2m was measured to be bioactive by the induction of alkaline phosphatase activity in MC3T3-E1 cells. Two hybridoma cell lines that stably secreted anti-rhBMP-2m antibody were developed from the immunized mice.</p><p><b>CONCLUSION</b>Bioactive rhBMP-2m protein and its monoclonal antibodies were successfully prepared, which will provides a solid base for future studies on rhBMP-2.</p>