克隆并构建人骨形成蛋白2真核表达载体

CLONING AND CONSTRUCTING OF BONE MORPHOGENETIC PROTEIN 2 EUKARYOTIC EXPERSSION VECTOR

目的探讨构建人骨形成蛋白2(humanbonemorphgeneticprotein2,hBMP-2)真核表达载体的方法。方法由人成骨肉瘤细胞中提取细胞总RNA,利用RT-PCR方法扩增获得hBMP-2基因cDNA,将基因片断重组到pGEM-T质粒中构建pGEM-T-hBMP-2重组质粒载体,转化到大肠杆菌DH5α后筛选阳性克隆,利用限制性酶切和DNA序列分析鉴定重组质粒。分别用EcoR和Not双酶切pGEM-T-hBMP-2质粒和pcDNA3.1(+)真核表达载体,将克隆载体中hBMP基因重组到pcDNA3.1(+)真核表达载体,提取质粒作酶切电泳、PCR鉴定及DNA测序。结果人骨肉瘤细胞中经RT-PCR得到1.2kbp的hBMP-2扩增条带,重组质粒pGEM-T-hBMP-2经酶切电泳可见1.2kbp的hBMP-2条带和4.0kbp的载体片断;pcDNA3.1-hBMP-2质粒经酶切电泳可见1.2kbp的hBMP-2条带和5.0kbp的载体片断,重组质粒酶谱分析与预期一致;DNA序列分析测序结果校对后经BLAST分析,表明核酸序列与GeneBank中和hBMP-2的mRNA序列完全吻合。结论通过此方法能成功克隆获得hBMP-2基因,并构建其真核表达载体。

Objective To clone human bone morphogenetic protein 2 （BMP-2） gene and construct the gene＇s eukaryotic expression vector. Methods The total RNA was extracted from human osteosarcoma cells, the human BMP-2 cDNA was amplified by RT PCR and inserted into pGEM-T vector. The positive clones were screened out, and then the recombinant plasmid was confirmed by restriction enzyme digestion, PCR and the analysis of nucleotide sequence. The BMP-2 cDNA in the pGEM T cloning vector was inserted into the pcDNA3.1 （＋） eukaryotic expression vector. Results The agarose electrophoresis showed that the fragments of BMP 2, pGEM T and pcDNA3. 1 （＋） were 1.2 kbp, 4.0 kbp and 5.0 kbp, respectively. The result of nucleotide sequence confirmed that the cDNA sequence, which was inserted into pGEM T and pcDNA3. 1 （＋） plasmid was human BMP-2. Conclusion The pcDNA3. 1 （＋）- hBMP-2 eukaryotic vector can be successfully constructed.