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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 explore a method to construct Ad-BMP-2 by using in vitro ligation instead of homologous recombination. METHODS: The BMP-2 gene was ligated into the helper vector pShuttle2 and digested with PI-Sce Ⅰ and Ⅰ-Ceu Ⅰ. The BMP-2 gene fragment containing the promoter Pcmvie was obtained and ligated to Recombinant adenovirus vector containing BMP-2 gene Ad-BMP-2 was obtained by double digestion of Adeno-X vector. After restriction endonuclease Pac Ⅰ digestion and linearization, the recombinant plasmid was transfected into HEK293 cells, and the recombinant adenovirus particles were packaged. The recombinant adenovirus was identified by PCR. Results: The recombinant vectors pShut-tle2-BMP-2 and Adneo-BMP-2 were identified by enzymatic digestion and PCR, respectively. The identification results were consistent with the theory. The packaged recombinant adenovirus was identified by PCR, and 1.2 kb and 287 bp bands were detected by 1% agarose electrophoresis, which was consistent with the size of the amplified fragment of MBP-2 and adenovirus. Conclusion: Compared with the traditional method of homologous recombination, the method of in vitro connection is simple and rapid, and this method does not require plaque purification, screening method is simple and convenient, and enhances the efficiency of adenovirus vector construction; at the same time, Ad-BMP.2 The successful construction laid the solid foundation for the subsequent research on the treatment of BMP-2 gene.