重组人骨形成蛋白-3成熟肽的表达、纯化及诱骨活性的研究

Expression,Purification and Bone inducing Activity of Recombinant Human Bone Morphorgenetic Protein 3 Mature PeptideKey Project of PLA's Medical Research Fundation(No.96Z045).

推测人骨形成蛋白３羧基端的１２７氨基酸的肽段为其成熟肽（ｈＢＭＰ３ｍ）。将编码此成熟肽的ｃＤＮＡ插入含ＰＬ启动子的表达质粒ｐＤＨ中，构建表达质粒ｐＤＨＢ３ｍ，转化大肠杆菌ＤＨ５α。４２℃热诱导６ｈ后表达量达到最高水平，约占菌体总蛋白２８％；表达产物以包涵体形式存在。包涵体经分离和洗涤后，溶解于尿素，在变性溶解状态下经阳离子交换层析，目的蛋白纯度至少在９５％以上。再经稀释复性。然后将纯化、复性的重组人骨形成蛋白３成熟肽（ｒｈＢＭＰ３ｍ）植入小鼠肌肉间隙，于不同时间取材观察，在局部可见典型的软骨形成、软骨内成骨以及骨组织形成的过程，证实所制备的ｒｈＢＭＰ３ｍ具有明显的异位诱骨活性。

It was inferred that the mature peptide of human bone morphorgenetic protein 3 (hBMP 3m) consisted of the carboxyl terminal 127 amino acid residues of hBMP 3.A plasmid,pDH B3m,which can express hBMP 3m,was constructed by inserting the cDNA sequences encoding hBMP 3m into pDH,a P L containing expression vector. Escherichia coli DH5α was transformed with pDH B3m.The highest expression level of recombinant hBMP 3m(rhBMP 3m) could be reached after 6h induction at 42℃,it amounted 28% of the total bacterial proteins.rhBMP 3m was in the inclusion body.After washing and paritally purification of the inclusion body,it was solubilized in urea and purified efficiently through ion exchange chromatiography.The purity of rhBMP 3m was at least 95%.rhBMP 3m was renatured by dilution method and then was implanted 1mg into mouse thigh muscles to assay its activity.A typical process of the formation of cartilage and bone tissues was observed from histological sections.The results showed that the purified and renatured rhBMP 3m had a good activity on inducing ectopic bone formation.