骨形态发生蛋白2真核表达载体的克隆与构建
[Cloning and constructing of bone morphogenetic protein 2 eukaryotic expression].
作者信息
Tian Xiaobing, Sun Li, Yang Shuhua
机构信息
Department of orthopedics, People's Hospital of Guizhou Province, Guiyang Guizhou 550002, P. R. China.
出版信息
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2006 Feb;20(2):112-5.
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.
目的
克隆人骨形态发生蛋白2（BMP-2）基因并构建该基因的真核表达载体。
方法
从人骨肉瘤细胞中提取总RNA，通过逆转录聚合酶链反应（RT-PCR）扩增人BMP-2 cDNA，并将其插入pGEM-T载体。筛选出阳性克隆，然后通过限制性内切酶消化、PCR及核苷酸序列分析对重组质粒进行鉴定。将pGEM-T克隆载体中的BMP-2 cDNA插入pcDNA3.1(+)真核表达载体。
结果
琼脂糖凝胶电泳显示，BMP-2、pGEM-T和pcDNA3.1(+)的片段大小分别为1.2 kb、4.0 kb和5.0 kb。核苷酸序列分析结果证实，插入pGEM-T和pcDNA3.1(+)质粒的cDNA序列为人BMP-2。
结论
可成功构建pcDNA3.1(+)-hBMP-2真核载体。
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