骨形态发生蛋白-2基因工程菌的构建及可溶性研究

Construction and soluble expression of recombinant Bone Morphogenetic Protein-2

骨形态发生蛋白是一类调节骨组织发育的生长因子,其中骨形态发生蛋白-2诱导成骨活性最强,在骨组织工程研究中最具研究意义.为获得能高效表达溶解性高的人骨形成蛋白-2(BMP-2)的基因工程菌,用PCR方法扩增得到BMP-2的基因序列,直接将PCR产物连接到胞内融合表达型T载体质粒pMT-L上,构建包括麦芽糖结合蛋白(MBP)、连接肽、6个His、EKsite(Asp-Asp-Asp-Asp-Lys)和BMP-2的表达载体,转化E.coli DH5α,经抗性筛选和菌落PCR鉴定,抽提阳性克隆质粒转化表达宿主E.coli BL21(DE3),成功构建可在大肠杆菌细胞质内表达MBP-BMP-2融合蛋白的基因工程菌.工程菌经0.1mmol/L IPTG诱导后,可获得表观分子量约为55kD的以可溶形式表达的BMP-2融合蛋白.

Bone morphogenetic proteins are a group of growth factors known for their ability to induce the formation of bone and cartilage. The bone morphogenetic protein-2 （BMP-2） displays a higher osteogenic activity than others, showing it has the most research significance in the bone tissue engineering. To obtain a high expression of soluble bone morphogenetic protein-2 （BMP-2） genetic engineering bacteria,the DNA sequence of Bone Morphogenetic Protein-2 （BMP-2） was amplified by polymerase chain reaction （PCR）. Then, the produce of PCR was directly recombined to the intracellular fusing expressional T-Vector pMT-L, constructing the expression plasmid that containing maltose-binding protein（MBP）, linker peptide, 6 His, EKsite （Asp-Asp- Asp-Asp-Lys） and BMP-2, then the recombinant plasmid transformed into E. coli DH5m After a drug-resistant selection and the identification by colonies PCR, the interested plasmid was extracted and transformed into E. coli BL21 （DE3）, the genetically engineered E. coli that can express MBP-BMP-2 fusion protein in the E. coli cytoplasm was successfully constructed. The results indicated that the genetically engineered bacteria expressed a large quantity soluble BMP-2 fusion protein in intracellular with an apparent molecular weight of about 55 kD when it was inducted by 0.1 mmol/L IPTG.