骨形态发生蛋白-7真核表达载体的构建及其在MC3T3-E1细胞中的表达

Construction of eukaryotic vector of bone morphogenetic protein-7 and its expression in MC3T3-E1 cells

目的构建一个能在MC3T3-E1细胞中表达骨形态发生蛋白-7(bmp-7)基因表达载体。方法采用RT-PCR技术从人胚肾中扩增人bmp-7基因,将获得的基因定向插入pcDNA3.1(+)真核表达质粒中,测序正确后用脂质体将表达质粒转染入MC3T3-E1细胞,转染后72h提取细胞全蛋白,采用Westernblot检测BMP-7蛋白表达。结果通过基因测序表明获得的bmp-7与GeneBank登录的序列一致,构建的质粒为bmp-7/pcDNA3.1(+)质粒,Westernblot检测结果表明bmp-7基因转染MC3T3-E1后能在细胞中表达。结论成功构建能在MC3T3-E1细胞中表达BMP-7的真核表达载体。

Objective To construct a eukaryotic vector which could express bone morphogenetic protein-7 （bmp- 7） in MC3T3-E1. Methods Bone morphogenetic protein-7 gene was obtained by RT-PCR from human embryo kidney. And after sequencing and electrophoresis the obtained aim DNA fragment was inserted into eukaryotic expression plasmid pcDNA3.1 （＋） by using restricted endonuclease and ligase. The DNA sequence of the newlyconstructed plamids was proved right by the gene technic company. And then the new plasmids containing right sequence aim gene were transfected into MC3T3-E1 cells by Lipofectamine 2000. 72 h after transfecting, RT-PCR was performed to show the transfected cells containing the aim gene, and the whole protein of the transfected cells were gathered and used as samples in the next Western blot to test the expression of bmp-7 gene. Results DNA sequencing indicated the sequence of the obtained bmp-7 was identical to the reported ones in GeneBank. The electrophoretic map of the products of RT-PCR and restriction enzyme digestion played another evidence that the newly-constructed plasmids were bmp-7/pcDNA3.1 （＋）. The results of Western blot showed that the transfected cells could express BMP-7. Conclusion The construction of a eukaryotic vector which could express BMP-7 in MC3T3- E1 was successful.