hOP-1全长基因克隆及真核表达载体的构建

目的证实人牙乳头细胞中OP-1的表达,获得hOP-1全长基因及其高效真核表达载体.方法提取人牙乳头细胞总RNA,反转录合成cDNA,以特异性引物扩增hOP-1全长基因并克隆入T载体,筛选阳性克隆进行序列测定后.构建高效真核表达载体pCI/OP-1并筛选鉴定.结果 PCR扩增得到一特异性的约1 300 bp的片段,序列测定结果表明与国外已发表的序列完全一致.构建的pCI/OP-1质粒,用于基因转染纯度较好.结论人牙乳头细胞中首次克隆到hOP-1全长基因,并构建获得该基因高效真核表达载体.

Cloning of hOP-1 full length gene and construction of its eukaryotic expression vector

Objective To clone the hOP 1 full length gene from human dental papilla mesenchymal cells for the construction of mammalian expression vector pCI/OP 1. Methods Total RNA was extracted from cultured human dental papilla mesenchymal cells, and the desired cDNA fragment was obtained by RT PCR with two specific gene primers. The fragment was inserted into T vector, and the resulted plasmid was transformed into DH5. The positive clone was selected and sequenced. Then the gene was subcloned into pCI to construct mammalian expression vector. Results The 1 300 bp specific fragment was obtained. The sequence of the hOP 1 full length gene was consistent with that of the references published. The purified pCI/OP 1 vector was good for gene transfection. Conclusion The hOP 1 full length gene has been cloned from human dental papilla mesenchymal cells for the first time, and the construction of its mammalian expression vector has been obtained. The results have provided basis for the further studies of the roles of OP 1 in tooth development as well as the functions of hOP 1.