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

Expression of Recombinant Mature Peptide of Human Bone Morphogenetic Protein-2 in Escharichia coil and Its Activity of Bone Formation

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.