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  • Title: [Construction of human bone morphogenetic protein-2 expressing eukaryotic vector].
    Author: Li XD, Hu YY, Pu Q.
    Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2001 May; 15(3):155-7. PubMed ID: 11393956.
    Abstract:
    OBJECTIVE: To construct human bone morphogenetic protein-2(hBMP-2) expressing eukaryotic vector and observe whether it can be expressed in eukaryotic cells. METHODS: pUC19 was digested with Sal I and Xba I. The resulting Sal I-Xba I fragment (1.24 kb) which contains the full length of human BMP-2 cDNA was separated on agarose gel and ligated into eukaryotic expression vector pcDNA3 digested with XhoI and XbaI. The recombinant pcDNA3-hBMP-2 plasmid was transferred into fibroblasts cell line NIH3T3. The stable expression of hBMP-2 in the positive cells G418 selected was determined by in situ hybridization and immunohistochemical analysis. RESULTS: The two fragments digested from recombinant pcDNA3-hBMP-2 plasmid by EcoR I and Xba I represented 1.3 kb and 5.38 kb respectively by agarose electrophoresis, meanwhile the Xho I site was disappeared in pcDNA3-hBMP-2 indicating the successful construction of recombinant pcDNA3-hBMP-2 plasmid. Stable expression of hBMP-2 in pcDNA3-hBMP-2 transfected cells was confirmed by in situ hybridization and immunohistochemical analysis. CONCLUSION: hBMP-2 expressing eukaryotic vector is successfully constructed and can be expressed in eukaryonic cells.
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