重组人BMP-2的基因克隆及原核表达

Cloning and Expression of Recombinant Human Bone Morphogenetic Protein-2 in E.Coli

目的构建重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2,rhBMP-2)的原核表达质粒并在大肠埃希菌中诱导表达。方法采用RT-PCR法,从骨髓细胞总RNA中扩增获得人BMP-2成熟肽cDNA,将其克隆入表达载体pBV220,构建BMP-2的重组原核表达质粒。重组质粒经酶切和测序鉴定后,在大肠埃希菌中诱导表达目的蛋白,表达产物采用Western印迹和ELISA进行鉴定。结果测序表明,重组基因序列与人BMP-2成熟肽基因完全一致。Western印迹和ELISA检测显示,表达产物的相对分子量(Mr)与预期结果相符,与相应抗体有结合活性。结论获得了人BMP-2成熟肽的编码基因,并构建了含有该基因的重组质粒pBV220/BMP-2,在大肠埃希菌中可高效表达人BMP-2。

Objective To construct a recombinant prokaryotie expression plasmid containg human bone morphogenetic protein-2 and express the protein in E. coli. Methods The mature peptide gene of human BMP-2 was amplified from human bone marrow and cloned into prokaryotic expression vector pBV220. The recombinant plasmid pBV220/BMP-2 was identified by DNA sequencing, before transfected into E. coli JM109. The recombinant protein expressed in E. coli was detected using western blot and ELISA. Results Sequencing analysis confirmed the sequence of recombinant BMP-2. The recombinant BMP2 protein was expressed with the right size as expected and displayed binding activity with its specific antibody. Conclusion The mature peptide gene of human BMP-2 has been successfully cloned and expressed in E. coli.