骨形态发生蛋白2表达载体的构建及生物学功能鉴定

Construction and Biological Function Characterization of BMP2 Expression Plasmid

目的:从真核细胞中获得骨形态发生蛋白2(BMP2)编码基因,构建其真核表达载体并进行鉴定。方法:提取人HeLa细胞总RNA,经反转录后用特异性引物扩增BMP2编码区,连接至pcDNA3.1载体,筛选阳性克隆并进行功能学鉴定。结果:反转录PCR获得1100 bp的片段,经分子克隆后鉴定序列与BMP2一致,并且在真核细胞中能够诱导Smad1/5/8的磷酸化。结论:构建了真核表达载体pcDNA3.1-BMP2并验证了其体内功能,为后续探讨BMP2在骨发育中的作用机制打下基础。

Objective： To clone bone morphogenetic protein 2（BMP2） full length gene from mammal cells, and then construct and characterize the mammal expression vector. Methods： The total RNA was extracted from HeLa cells. Amplify BMP2 full length cDNA through RT-PCR with a pair of specific gene primers. Clone it into pcD- NA3.1 vector. The positive clone was selected and sequenced. Verify the biological function of BMP2 in mammal cells. Results： The 1100 bp specific fragment was obtained by RT-PCR. After the molecular cloning and sequencing, BMP2 over-expressed in mammal cells can induce the phosphorylation of Smadl/5/8. Conclusion： We constueted pcDNA3.1-BMP2 and confirmed its function. These data provide a basis of the mechanistic study of BMP2 function in bone development.