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重组人骨形态发生蛋白—2成熟肽在大肠杆菌中的表达及其诱导成骨活性 被引量:76

Expression of Recombinant Mature Peptide of Human Bone Morphogenetic Protein-2 in Escharichia coil and Its Activity of Bone Formation
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摘要 报告了人骨形态发生蛋白-2(BMP-2)成熟肽的基因克隆,及其在大肠杆菌中的表达。用AflⅡ酶剪除BMP-2cDNA中的信号肽及前肽部分的序列,将编码成熟肽的cDNA3′端0.36kb基因片段克隆于大肠杆菌表达载体pMX-1质粒的ATG下游,受控于PLPR启动子。重组子以大肠杆菌DH5α为宿主细胞进行温度诱导表达。工程菌经42℃6h诱导后,在SDS-PAGE上出现一条新蛋白带,分子量约为12kD,约占菌体总蛋白的20%。主要以包涵体形式存在的表达产物经初步纯化后,可获得纯度较高的重组人骨形态发生蛋白-2成熟肽(rhBMP-2m)。小鼠肌肉植入试验发现。rhBMP-2m植入后的不同时间,在局部出现间质细胞的聚集和增生、软骨细胞及软骨基质的生成和硬质骨的形成,证明rhBMP-2m具有明显的诱导新骨形成的作用。 Human bone morphogenetic protein-2(hBMP-2) consists of signal peptide, propetide and mature peptide. The 0. 36kb DNA fragment encoding mature peptide were obtained with Afl Ⅱ and EcoR Ⅰ from 3' termini of hBMP-2 cDNA. The fragment was inserted into expression vector pMX-1 following ATG that was controlled by P_LP_R promoter. The recombinant plasmid pMB-2m was transformed into E. coli DH5α and induced at 42℃ to express encoded proteins. SDS-PAGE revealed a new foreign protein band near 12kD after 6h induction. The recombinant muture peptide of hBMP-2 (rhBMP-2m) in inclusion bodies made up 20% of the total bacteria proteins and was purified partially. Bioassay of implantation of mouse thigh showed that rhBMP-2m induced the proliferation and migration of mesenchymal type cells which then differentiated into chondrocyte and cartilage, and finally formed new bone in the implantation area.
作者 赵明 王会信 Zhao, Ming; Wang,Hui-xing; Zhou,Ting-chong(Institute of the Basic Medical Sciences, Academy of Medical Sciences, Beijing 100850)
出处 《生物化学杂志》 1994年第3期 319-324,共6页
关键词 骨形态发生 蛋白 DNA 重组 表达 Human bone morphogenetic proyein-2 (hBMP-2 ) Recombinant DNA Gene expression Inclusion body Cartilage Bone formation
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