Construction of Eukaryotic Expression Vector of Bone Morphogenetic Protein Gene and Its Expression in Chinese Hamster Ovarian Cells

【Objective】To clone human bone morphogenetic protein 4(BMP4) gene, construct BMP4 eukaryotic expression vector and observe its expression in Chinese hamster ovarian(CHO) cells,thus to provide evidence for the further study of gene therapy for bone repair.【Methods】The amplified cDNA of human BMP4 from cancellous bone was obtained by reverse transcription polymerase chain reaction(RT-PCR) and then was inserted into pGEM-Teasy cloning vector.After the sequence of pGEM-Teasy vector including amplified human BMP4 cDNA was confirmed,BMP4 eukaryotic expression vector was constructed from its subclone with PCDNA3.1B(-).Then the recombinant plasmid of PCDNA3.1B(-) BMP4 was transfected into CHO cells through lipofectamine method and positive cellular clones were screened with G418.Human BMP4 gene expression in the transfected cells was confirmed with RT-PCR and immunocytochemistry.【Results】Restriction enzyme digestion and DNA sequencing analysis showed that the target gene was inserted into the recombinant vector.The results of RT-PCR and immunocytochemistry showed the expression of human BPM4 gene in transfected CHO cells.【Conclusion】The full-length of human BMP4 is obtained successfully and its sequence is highly consistent with the known sequence.