可调控表达人骨形态发生蛋白2基因的腺病毒载体系统的构建

目的构建可调控人骨形态发生蛋白2（hBMP2）基因表达的重组腺病毒载体系统，为骨缺损的修复提供新思路。方法基因测序验证pcDNA3．1－hBMP2质粒的准确性，将酶切得到的hBMP2基因片段亚克隆到穿梭载体质粒pTRE—Shuttle2的多克隆位点，将pTRE—Shuttle2-hBMP2线性化并连接至腺病毒骨架质粒Adeno—XSystem 1 Viral DNA上，构建重组的Adeno—X－pTRE—hBMP2载体；用PacI酶将其线性化后脂质体法（Lipofectamine 2000）转染HEK293细胞，获得有感染能力的重组腺病毒颗粒，收集病毒上清进行hBMP2基因PCR鉴定；将包含rtTA激活因子的Adeno—X Tet—Onvirus Stock感染HEK293细胞包装扩增病毒。结果hBMP2基因被成功定向克隆到腺病毒骨架质粒Adeno—X System 1 Viral DNA上，转染HEK293细胞后获得有感染能力的重组腺病毒颗粒。结论本研究成功构建了hBMP2基因的腺病毒Tet—On表达系统，为实现hBMP2基因在靶细胞水平上的调控表达提供前期实验基础。

Construction of a Tetracycline-regulated recombinant adenovirus vector expressing hBMP2

Objective To construct a Tetracycline-regulated recombinant adenovirus vector expressing human bone morphogenetic protein 2 （hBMP2） and to provide a new method for repairing bone defect. Methods The peDNA 3.1-hBMP2 plasmid was verified by gene sequence analysis. The hBMP＇2 eDNA was sub-cloned into the pTRE-Shuttle 2 vector by molecular cloning and then the recombinant pTRE-Shuttle 2-hBMP2 linear DNA fragments were cloned into Adeno-X System 1 Viral DNA. The recombinant Adeno-X-pTRE-hBMP2 was digested by Pac I and then transfected to the HEK 293 by lipofectamine 2000 mediated gene transfer. The hBMP2 gen was identified by PCR method. At the same time Adeno-X Tet-On virus Stock was transfected to the HEK293 by the same method to get infective virus particles. Results hBMP2 gene was cloned into Adeno-X System 1 Viral DNA successfully and the infective virus particles were obtained. Conclusions An Tet-On expression vector for transferring hBMP2 gene into adenovirus was constructed successfully and will make a help for bone defection.