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构建重组人骨形态发生蛋白-2/7融合体, 研究其在大肠杆菌中的表达及生物学活性。利用DNA 重组技术,将编码人骨形态发生蛋白-2 (BMP-2) 成熟肽的0.36 kb 基因片段与编码人骨形态发生蛋白-7 (BMP-7) 成熟肽的0.44kb 基因片段连接,得到重组人骨形态发生蛋白-2/7 融合基因; 经测序鉴定后,将其克隆到表达载体pBV222中,转化大肠杆菌DH5α, 温度诱导表达。表达蛋白经初步纯化后测定其对培养小鼠成骨样细胞增殖、分化及碱性磷酸酶活性的影响。克隆质粒转化的工程菌,经42℃诱导4~6 h 后,高效表达重组人骨形态发生蛋白-2/7, 在SDS-PAGE上出现一新的蛋白带, 分子量约29 ku,约占菌体总蛋白的20% ,表达蛋白以包涵体的形式存在,经初步纯化、裂解、复性后的BMP-2/7 蛋白具有促进成骨样细胞分化、增殖及增加碱性磷酸酶活性的作用。BMP-2/7 融合基因的构建和表达,可能提供一种全新的具有诱导成骨作用的蛋白
Recombinant human bone morphogenetic protein -2/7 fusion was constructed and its expression in E. coli and its biological activity were studied. The 0.36 kb gene fragment encoding human bone morphogenetic protein-2 (BMP-2) mature peptide was ligated with the 0.44 kb gene fragment encoding mature peptide of human bone morphogenetic protein-7 (BMP-7) by DNA recombination technology. Recombinant human bone morphogenetic protein-2/7 fusion gene was obtained. After sequencing, it was cloned into expression vector pBV222 and transformed into E. coli DH5α for expression under the temperature. The expressed protein was preliminarily purified and its effect on proliferation, differentiation and alkaline phosphatase activity of cultured osteoblast-like cells were determined. Cloning plasmid transformed engineering bacteria, after 42 ° C induction of 4 ~ 6 h, recombinant human bone morphogenetic protein-2/7 highly expressed in SDS-PAGE appeared a new protein band, molecular weight of about 29 ku, accounting for about bacteria BMP-2/7 protein after primary purification, lysis and refolding can promote differentiation, proliferation and increase alkaline phosphatase activity of osteoblast-like cells. The construction and expression of the BMP-2/7 fusion gene may provide a brand new protein with induced osteogenesis