目的：探索采用体外连接技术替代同源重组方法构建Ad-BMP-2，优化腺病毒载体构建方法。方法：将BMP-2基因连接到辅助载体pShuttle2后，以限制性内切酶PI-SceⅠ及Ⅰ-Ceu Ⅰ酶切，获得含启动子Pcmvie的BMP-2基因片段，然后将其连接到经同样双酶切的Adeno-X载体上，获得含BMP-2基因的重组腺病毒载体Ad-BMP-2。将其以限制性内切酶Pac Ⅰ酶切线性化后，转染HEK293细胞，包装重组腺病毒颗粒，并采用PCR方法对重组腺病毒进行鉴定。结果：重组载体pShuttle2-BMP-2及Adneo-BMP-2均采用酶法切及PCR鉴定，鉴定结果均与理论相符。将包装后的重组腺病毒以PCR鉴定，以1％琼脂糖电泳见1.2 kb及287 bp有带，与MBP-2及腺病毒扩增片段大小相符。结论：与传统的同源重组方法相比，体外连接技术方法简单、快捷，并且此方法不需噬斑纯化、筛选方法简便，提高了腺病毒载体构建的效率；同时，Ad-BMP-2的构建成功为后续进行BMP-2基因的治疗研究奠定了坚实的基础。

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.

国家自然科学基金资助项目(30271349)