重组人骨形成蛋白-3羧基端肽在大肠杆菌中表达及其诱骨活性

以双顺反子表达载体，在大肠杆菌中经ＩＰＴＧ诱导表达了人骨形成蛋白－３羧基端肽段（ｈＢＭＰ－３Ｃ），表达量占菌体总蛋白量的１８．５％．目的蛋白为２５ｋＤ、含ｈＢＭＰ－３Ｃ端２１５个氨基酸残基组成的肽段，包括ｈＢＭＰ－３成熟肽和一部分前肽．表达产物以包涵体的形式存在，用含ＴｒｉｔｏｎＸ－１００的洗涤液和５ｍｏｌ／Ｌ以下脲溶液连续洗涤，可获得较高纯度的重组人骨形成蛋白－３Ｃ端肽．经复性处理成可溶性蛋白，植入小鼠肌肉内，第１４ｄ组织切片显示有软骨细胞和软骨基质形成，第２１ｄ可见成骨细胞和骨基质形成．将ｒｈＢＭＰ－３Ｃ与脱矿去免疫原性异种骨粒复合后作小鼠肌肉植入试验，２１ｄ组织切片上可见硬质骨形成．结果表明：大肠杆菌表达的ｈＢＭＰ－３Ｃ经复性后具有诱骨活性，糖基化并非ＢＭＰ－３活性所必需．

Bone Induction Activity of Carboxyl Terminal Peptide of Human Bone Morphogenetic Protein 3 Expressed in Escherichia coli

A cDNA fragment encoding the carboxyl terminal 215 amino acids of human bone morphogenetic protein 3 (hBMP 3C) was subcloned into a two cistron expression vector,in which contains a truncated GST gene as the first cistron.After transforming in E.coli JM109 and inducing with IPTG,hBMP 3C was expressed in inclusion bodies and amounted to 18 5% of the total bacterial proteins.After partial purification and renaturation of the product,1 mg rhBMP 3C was implanted alone into mouse muscles.Chrondroid and osteoid structure were found in tissue sections at 14 and 21 days.On the other way, rhBMP 3 adsorbed with a carrier particle which prepared from a demineralized bovine bone,removed off immunity,was implanted into mouse muscle.The formation of mature bone tissue was observed at 21 days.The results suggested that the renatured rhBMP 3C expressed in E.coli had good bone induction activity and glycosylation of BMP 3 was not essential for its activities.