人骨形成蛋白-2基因腺病毒表达载体构建方法的优化

Construction of Recombinant Adenoviral Vector Carrying the Human BMP-2 Gene with the Technology of in vitro Ligation

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—Sce Ⅰ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pShut—tle2-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-SCe Ⅰ and Ⅰ-Ceu Ⅰ endonuclease. The fragment was further combined with adenovirus vector (Adeno-X-BMP-2) , which was finally linearized with Pac Ⅰ and trans-fered 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 i-dentified 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 de-scinbed here for constructing recombined adenovirus was more efficient than traditional homologous recombination.