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Published on: October 30, 2018
[Construction of eukaryotic vector of bone morpbogenetic protein-7 and its expression in MC3T3-E1 cells]
1Dental Clinic, Sir Run Run Shaw Hospital Affiliated to Medical College of Zhejiang University, Hangzhou 310016, China.
Objective:
To construct a eukaryotic vector which could express bone morphogenetic protein-7 (bmp-7) in MC3T3-E1.
Methods:
Bone morphogenetic protein-7 gene was obtained by RT-PCR from human embryo kidney. And after sequencing and electrophoresis the obtained aim DNA fragment was inserted into eukaryotic expression plasmid pcDNA3.1 (+) by using restricted endonuclease and ligase. The DNA sequence of the newly-constructed plamids was proved right by the gene technic company. And then the new plasmids containing right sequence aim gene were transfected into MC3T3-E1 cells by Lipofectamine 2000. 72 h after transfecting, RT-PCR was performed to show the transfected cells containing the aim gene, and the whole protein of the transfected cells were gathered and used as samples in the next Western blot to test the expression of bmp-7 gene.
Results:
DNA sequencing indicated the sequence of the obtained bmp-7 was identical to the reported ones in GeneBank. The electrophoretic map of the products of RT-PCR and restriction enzyme digestion played another evidence that the newly-constructed plasmids were bmp-7/pcDNA3.1(+). The results of Western blot showed that the transfected cells could express BMP-7.
Conclusion:
The construction of a eukaryotic vector which could express BMP-7 in MC3T3-E1 was successful.
