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Construction of Recombinant Adenoviral Vector Carrying the Human BMP-2 Gene with the Technology of in vitro Ligation
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Abstract:
Objective: To provide an efficeut protocol for constructing recombinant adenovirus, an in vitro ligation was used instead of homologous recombination. Methods: Gene BMP-2 was ligated into pShuttle 2 vector (pShuttle 2-BMP-2) and then fragment containing BMP-2 gene and promoter pcmvie excised by PI-SCeⅠ and I-CeuⅠ endonuclease. The fragment was further combined with adenovirus vector (Adeno-X-BMP-2), which was finally linearized with PacⅠ and transfered to HEK293 to package adenovirus particles. Results: Both PCR assay and restiction analysis showed that the recombined rectors pShuttle2-BMP-2 and Adeno-X-BMP-2 contains the target BMP-2 gene. THe packaged adenovirus was also identified by PCR assay with specific primers for BMP-2. Conclusions: The BMP-2 incorporated recombinant adenovirus was obtained and this laid a foundation for further study on BMP-2 mediated gene therapy. The in vitro ligation method descinbed here for constructing recombined adenovirus was more efficient than traditional homologous recombination.
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