BMP-4cDNA的克隆表达及活性的初步测定

目的：利用原核基因工程生产有诱骨活性的骨形态发生蛋白－4（BMP－4），方法：应用RT－PCR技术，从成熟人的胎盘组织中扩增出长0．34Kb编码入骨形态发生蛋白－4成熟肽的基因序列，经测序证实后，装入表达载体pET-22b，诱导表达后SDS－PAGE显示在14kd处有一明显的表达条带，经初步鉴定，证实表达蛋白部分位于裂菌处理后的上清中，部分以包涵体的形式存在于沉淀中，将沉淀中的包涵体蛋白纯化，变性和复性处理，和上清中的蛋白分别经真空冷冻干燥后，植入小鼠的肌肉内检测蛋白活性。结论：第14d和21d，组织切片可见骨细胞和骨基质的形成，结论：大肠杆菌表达的BMP－4的经复性处理后具有诱骨活性。

Cloning and Expression of Mature Peptide of Human Morphogenetic Protein-4cDNA in Escharichia Coil and Bioassay in vivo

Objective To produce the bioactive bone morphogenetic protein-4(BMP-4) by the way of genetic engineering. Methods cDNA of human morphogenetic protein-4 mature peptide was obtained by RT-PCR from the tissue of human placenta. The gene was constructed into the pET-22b(+) expression vector and expression in Escharichia coil BL-21 after transformation and induction by IPTG. SDS-PAGE revealed a new protein band that located in position of 14kd after 4h induction, the new protein made 15% of total bacteria protein. It was identified to dissolve partially in the supernatant through the way of lysating bacteria, another part was expressed in the precipitation as the way of inclusion body. The inclusion body was purified by urea solution of different concentration and denaturalized, naturalized. The protein was harvested by vacuum freeze machine and was implanted in the mouse thigh. Results Bioassay of implantation in mouse thigh showed that rhBMP-4 induced the proliferation and migration of mesenchymal cells, and then differentiated into chondrocyte and cartilage. Finally fromed new bone in the implantation area. Conclusion The expressed BMP-4 of Escharichia coil BL-21 can have bioactivity by the way of naturalized.