人骨形成蛋白2碳端肽在大肠杆菌中的高表达及活性测定

C terminal peptide of hBMP2:high expression in E.coli and boneinductive activity

目的：利用基因工程技术生产人骨形成蛋白（ｈＢＭＰ），为其诱骨机理及临床应用研究提供前提条件。方法：作者将ｈＢＭＰ２ｃＤＮＡ３’端７３２ｂｐ片段，克隆入大肠杆菌表达载体ｐＧＥＸ－２Ｔ，经ＩＰＴＧ诱导后，表达产物存在于包涵体中。将其纯化复性后，进行ＳＤＳ－ＰＡＧＥ和ＦＰＬＣ分析。并作小鼠体内异位诱骨活性检测。结果：表达的ｈＢＭＰ２与ＧＳＴ的融合蛋白Ｍｒ＝５１ｋｕ，占菌体总蛋白的３０％。纯化、复性后，ＦＰＬＣ分析示尖而窄的洗脱峰。ＳＤＳ－ＰＡＧＥ呈一条带。在小鼠肌肉中可诱导软骨及骨组织生成。结论：ｈＢＭＰ２在大肠杆菌中的表达产物复性后纯度较高，且具有诱骨活性。

Objective:By way of genetic engineering production of human bone morphogenetic protein(hBMP2),it will offer the basis for the clarification of its bone inductive mechanism and clinical application. Methods:3’terminal fragment of hBMP2 cDNA with size of 732 bp was cloned into pGEX+2T expressionvector and induced by IPTG,the expressed product existed in a form of inclusion body.After purification andrenaturation of rhBMP2,its bone inductive activity was assayed in mouse heterotopically in vivo. Results:The fusion protein of GST with hBMP2 is 51 ku in size. After purification and renaturation,FPLC analysisshowed a sharp and narrow elution peak.The result on SDS-PAGE appeared in vivo one band.The histolog-ic assay displayed that rhBMP2 can induce the formation of cartilage and bone tissue. Conclusion:TherhBMP2 expressed in E,coli was able to induce bone formation after renaturation of inclusion body. The re-natured protein had a relatively high purity.