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

作　　者：王婧[1] 张杰 张颖 马宁宁[1] 谢良志[1] 

机构地区：[1]中国医学科学院北京协和医学院细胞工程中心,北京100005 [2]神州细胞工程有限公司,北京100176

出　　处：《中国医学科学院学报》2011年第5期543-548,共6页Acta Academiae Medicinae Sinicae

基　　金：十一五"重大新药创制"科技重大专项(2009ZX090306-004)~~

摘　　要：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.

关 键 词：重组人骨形态发生蛋白-2成熟肽 蛋白复性 单克隆抗体 

分 类 号：Q861[生物学]