人骨形成蛋白-2C端102肽的诱骨活性

为分析更短的Hbmp-2C端肽是否具有诱骨活性 ,寻求新型的有诱骨活性的基因工程Hbmp-2产品。利用温度诱导的大肠杆菌表达系统表达肽段长度为102个氨基酸的Hbmp-2C端肽及其Cys的突变体。表达产物经纯化复性后 ,植入小鼠肌袋模型中测试其诱骨活性。获得了能稳定表达Hbmp 2C端肽的工程菌 ,测序结果与预期的序列完全一致。表达产物以包涵体形式存在 ,表达量占细胞总蛋白的 3 0 %。产物经纯化复性后 ,小鼠肌袋模型测试结果表明 :Hbmp 2 10 2肽仍具有诱骨活性 ,而将C端第一位Cys突变的 10 2肽诱骨活性丧失。实验表明 :比Hbmp 2成熟肽 ( 114个氨基酸 )更短的C端 10 2肽仍具有良好诱骨活性 ,这 10 2肽N端第一个Cys对其诱骨活性可能是必需的。

Bone-Inducing Activity of Human Bone Morphorgenetic Protein-2 102 Peptide

To analyze the bone-inducing activity of C terminal of hBMP-2 and get a new recombinant product of hBMP-2, the gene encoding 102 aa of hBMP-2 mature peptide C terminal was cloned and expressed in E. coli and the first Cys was mutated with Ser. The fragments encoding the target peptide were amplified and cloned into heat-inducible expression vector pDH and transformed into E. coli DH5 alpha. After induction, a new protein bond appeared on the SDS-PAGE. The expressed products amounted to 30% of the total bacterial protein, which existed in the form of inclusion body. The products of bacterial lysates were purified through the ion-exchange chromatography. The denatured proteins were dialysed and diluted directly into the refolding buffer. The renatured products were implanted into mouse thigh muscles to analyze their bone-inducing activity respectively. The results of histological assay showed that the 102 peptide of hBMP-2 could ectopically induce formation of bone, while the mutated 102 peptide of hBMP-2 could not. It suggested hBMP-2 102 peptide still had bone-inducing activity. The first Cys of hBMP-2 mature peptide might be necessary for integrity of three pairs of disulfide bond, and also essential for bone-inducing activity of hBMP-2.