重组人骨形态发生蛋白-2原核表达及单克隆抗体制备

Prokaryotic Expression of Recombinant Human Bone Morphogenetic Protein-2 and Preparation of Monoclonal Antibodies

目的利用原核表达系统(E.coli)表达纯化重组人骨形态发生蛋白-2成熟肽(rhBMP-2m),并制备rh-BMP-2m的单克隆抗体。方法将工程菌株进行常规发酵,自诱导表达,裂菌离心分离包涵体,包涵体变性后经阳离子交换色谱分离纯化。纯化后的变性rhBMP-2m经稀释复性,获得具有生物学活性的rhBMP-2m。并以此作为抗原,免疫Balb/c小鼠制备单克隆抗体。结果获得还原SDS-PAGE下95%以上纯度的rhBMP-2m。细胞活性结果分析显示,所获得的rhBMP-2m具有较强的生物学活性。免疫小鼠最终获得两株稳定分泌抗rhBMP-2m抗体的杂交瘤细胞株。结论成功制备了具有生物学活性的rhBMP-2m及其单克隆抗体,为rhBMP-2未来的研究和制备奠定良好的基础。

Objective 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.Methods 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 cantion 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.Results 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.Conclusion Bioactive rhBMP-2m protein and its monocloncal antibodies were successfully prepared,which will provides a solid base for future studies on rhBMP-2.