The Experimental Research Of The Construction Of The Recombinant Adenovirus Carring The Human Bone Morphogenetic Protein-2 Gene

Objection: To constuct recombinant adenovirus carring the human bone morphogenetic protein-2(hBMP2) gene and amplify the adenovirus vector in 293E cells for gene therapy, gene modification to encourage the development of osteoblasts from mesenchymal stem cells.Methods: First, routining CaCl₂ method was used to transform pcDNA3 plasmid carring hBMP2 gene into chemical compenent E. coli DH5a cells. Then, pcDNA3-hBMP2 plasmid was extracted from E. coli DH5a cells using the Rapid Plasmid DNA Daily-prep Kit( V-gene). Consequently, a insert from the plasmid was released by the double digestion with the restriction endonucleases KpnI and XhoI. Then, the pcDNA3-hBMP2 plasmid was identified as anastomose with the reported hBMP2 gene by sequencing and accordant with the pcDNA3-hBMP2 plasmid map using 1% agarose gel electrophoresis.Second, the adenoviral shuttle vectors(pAdTrack-CMV) was transformed into chemical compenent E. coli DH5a cells. Then, after the extraction from those cells by the Rapid Plasmid DNA Daily-prep Kit( V-gene), pAdTrack-CMV plasmid was digested with the restriction endonucleases KpnI, PacI and XhoI. The productions of DNA fragments separated by 1.0% agarose gel electrophoresis were the same with the predictive outcome from the multiple cloning restriction sites noted on the pAdTrack-CMV plasmid map.Third, pcDNA3-hBMP2 plasmid and pAdtrack-CMV plasmid were digested by the combination of the restriction endonucleases KpnI and XhoI and separated by 1% agarose gel electrophoresis. After that, the fragments of hBMP2 and pAdtrack-CMV vector were recovered from agarose gel by the DNA Gel extraction kits(V-gene).Then, the fragment of hBMP2 gene was inserted into the pAdtrackCMV vector fragment in order to generate a recombinant pAdTrackCMV-hBMP2. The outcome of pAdTrackCMV-hBMP2 plasmid digested with the restriction endonucleases Kpnl, Pad and Xhol was identified to be the same as the anticipated fragments using 1% agarose gel electrophoresis, which suggested that the recombinant pAdTrackCMV-hBMP2 plasmid was recombined correctly.Fourth, E. coli BJ5183 electrocompenent cells were prepaired according to Gene Pulser Xcell? instruction manual and the pAdEasey-1 plasmid was electroporated into E. coli BJ5183 cells. Then, the pAdEasey-1 plasmid was extracted from E. coli BJ5183 cells used the Rapid Plasmid DNA Daily-prep Kit( V-gene). A single colony containing pAdEasey-1 was subsequently characterized by the restriction endonucleases Kpnl, Pad and Xhol analysis and was named as AdEasier-1 cells. AdEasier-1 eleetrocompenent cells was prepared using the same method as upper.Fifth, the Pmel-linearized pAdtrackCMV-hBMP2 plasmid, without enzyme inactivation and purification, was directly electroporated into AdEasier-1 electrocompenent cells. The recombinant plasmid was obtained by homologous recombination in E. coli BJ5183 cells and was extracted using the Rapid Plasmid DNA Daily-prep Kit( V-gene). Further more, the recombinant plasmid pAd-hBMP2 was tested by o.7% agarose gel electrophoresis after the digestion with the restriction endonuclease BamHI and Pad. Then pAd-hBMP2 plasmid was transformed into E. coli DH5a cells for large-scale amplification by electroporation.Last, a transfection mix was prepared by adding 4 //g of linearized pAd-hBMP2 plasmid DNA with Pad and 20 [A of transfection reagent (Sofast?) to 500 jul of IMDM according to the manufacturer's instructions. Following that, the recombinant adenovirus was identificated by means of observation of green fluorescence protein expression under fluorescent microscope four days later. After amplified in 293E cells, the Adenovirus was transfected into 293 cells, and then, the green fluorescence protein expression was detected.Results: The recombinant adenovirus vector earring hBMP2 gene was constructed successfully. Besides, the recombinant virus was obtained at high titer, which could express with high efficiency in vivo.Conclusions: The recombinant adenovirus vector earring hBMP2 gene was constructed successfully, which would have some basal effect on the further research of the gene therpy using hBMP2 gene.