Value of construction of bone morphogenetic protein-2 recombinant adenovirus on genetic treatment of bone defect / 中国组织工程研究

ABSTRACT

BACKGROUND: At present, genetic recombinant technique has been utilized to express recombinant human bone morphogenetic protein-2 (rhBMP-2)and to induce either orthotopic or ectopic regenerated bone successfully. But,because that osteoblastic activity induced by rhBMP-2 is lower than that by natural BMP-2, the perfect vectors have not been discovered yet.OBJECTIVE: BMP recombinant adenovirus (rAdV) was constructed so as to provide feasible vector for the basic treatment of bone defect.DESIGN: Single sample was designed in the experiment.SETTING: Experimental Center of Molecular Biology in First Hospital of Xi'an Jiaotong University.MATERIALS PACCMV-PLPA plasmid, PJM17 plasmid, 293-cell line.METHODS: The experiment was performed in Experimental Center of Molecular Biology in First Hospital of Xi'an Jiaotong University from September 2001 to June 2002. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to clone the whole-length gene of BMP-2 and construct AdV vector. DNA-calcium phosphate coprecipitation was used to transfect accessory plasmid PJM17 into 293cell and homologous recombination was used to construct rAdV. The titer was assayed and CsC1 density gradient centrifugation and purification were applied.construction of rAdV.binant plasmid is named as pGEM-T/hBMP2, (3 015+1 213)bp in size.It was indicated in agar gel electrophoresis that the practical value was in BMP2 shuttle plasmid The plasmid was about 10 Kbp in size. Since PACCMV-PLPA plasmid multiclone sites belonged to PUC plasmid series,EcoR Ⅰ enzyme digestion was used to linerarizate recombinant plasmid,10 Kbp in size. 8.8 Kb and 1.2 Kbp visible fragments were obtained durtion of rAdV PCR technique was used to identify rAdV. The target fragment 1.2Kbp that was amplified by BMP2 specific primer of the whole length was in conformity completely with the theoretic value.CONCLUSION: Successful construction of BMP2 rAdV lays a foundation for the feasible genetic treatment with vector of bone defect.