人骨形态发生蛋白—2真核表达载体的构建

目的 构建人骨形态发生蛋白－2（hBMP-2）真核表达载体，并观察其在真核细胞内表达的可能性。方法 Sal Ⅰ和Xba Ⅰ双酶切含有hBMP－2全长cDNA的pUC19，回收1.24kb片段，将其连入真核表达载体pcDNA3，构建重组子pcDNA3-hBMP-2。将重组子转化细菌，扩增后提取质粒，分别用Ecor Ⅰ和Xba Ⅰ双酶切和Xho Ⅰ单酶切后琼脂糖凝胶电泳鉴定。将重组子转染成纤维细胞，G418筛选形成阳性克隆后继续培养4周行细胞原位杂交和免疫组织化学染色。结果 琼脂糖电泳显示：用EcoR Ⅰ和Xba Ⅰ双酶切后可形成两条带，大小分别为1.3kb和5.38kb，而Xbo Ⅰ酶切位点消失，符合物理图谱，表明载体构建成功，转染后G418筛选所获阳性克隆，继续培养4周，的位杂交和免疫组织化学染色，结果表明hBMP-1基因在mRNA水平和蛋白水平均有表达。结论 成功构建hBMP-2真核表达载体，该载体可在真核细胞内表达，为将来骨损伤的基因治疗奠定了一定的实验基础。

CONSTRUCTION OF HUMAN BONE MORPHOGENETIC PROTEIN-2 EXPRESSING EUKARYOTIC VECTOR

Objective To construct human bone morphogenetic protein 2(hBMP 2) expressing eukaryotic vector and observe whether it can be expressed in eukaryotic cells. Methods pUC19 was digested with Sal I and Xba I. The resulting Sal I Xba I fragment (1.24 kb) which contains the full length of human BMP 2 cDNA was separated on agarose gel and ligated into eukaryotic expression vector pcDNA3 digested with XhoI and XbaI. The recombinant pcDNA3 hBMP 2 plasmid was transferred into fibroblasts cell line NIH3T3. The stable expression of hBMP 2 in the positive cells G418 selected was determined by in situ hybridization and immunohistochemical analysis. Results The two fragments digested from recombinant pcDNA3 hBMP 2 plasmid by EcoR I and Xba I represented 1.3 kb and 5.38 kb respectively by agarose electrophoresis, meanwhile the Xho I site was disappeared in pcDNA3 hBMP 2 indicating the successful construction of recombinant pcDNA3 hBMP 2 plasmid. Stable expression of hBMP 2 in pcDNA3 hBMP 2 transfected cells was confirmed by in situ hybridization and immunohistochemical analysis. Conclusion hBMP 2 expressing eukaryotic vector is successfully constructed and can be expressed in eukaryonic cells.