【摘要】 目的:采用基于attLXattR的λ噬菌体位点特异性重组系统的Gateway^TM技术构建人骨形态发生蛋白-2基因重组腺病毒载体（Ad-hBMP-2）。方法:从pcDNA3.1质粒中获取目的基因hBMP-2片段,连入带attL1、attL2位点的入门载体pENTRTM11中,形成入门克隆,将入门克隆与带attR1、attR2位点的目的载体Ad/CMV/V5-DEST在高效重组酶作用下发生体外重组形成表达克隆ad-BMP-2,经鉴定将ad-BMP-2线性化后转入293A细胞包装,通过细胞裂解法获得人骨形态发生蛋白-2的基因重组腺病毒珠Ad-hBMP-2,将其扩增,采用western blotting技术分析目的蛋白表达。结果:经酶切及测序证实目的基因BMP-2片段按正确方向重组入目的载体中,带BMP-2的目的载体在293A细胞中包装成功,获得成熟的病毒颗粒,测得病毒滴度为2.5×10⁹pfu/ml,western blotting结果证实Ad-hBMP-2在293A细胞中高效表达hBMP-2蛋白,结论:本实验首次利用基于λ噬菌体的位点特异性重组系统的Gateway^TM技术成功构建了Ad-hBMP-2,该技术与传统构建方法比较具有高效性和灵活性,为进一步研究BMP-2的生理功能和利用BMP-2进行骨基因治疗奠定了实验基础。更多还原

【Abstract】 Objective:To establish a recombinant adenovirus vector with BMP-2 byλphasge-site specific recombination systems. Methods:The target gene of hBMP-2 fragment was prepared from plasmid pcDNA3.1 and ligated into entry vector pENTRTM11 with attL1 and attL2 sites to generate the entry clone.Then the entry clone and the target vector Ad/CMV/V5-DEST with attL1 and attL2 sites was recombined together in vivo to create the expression clone（ad-BMP-2）LR under the action of the efficient recombines.After the expression clone confirmed by restriction analysis,PCR and sequencing,ad-BMP-2 was digested with Pac（?）,and transferred into 293A cells to be packaged into adenovirus stock（Ad-hBMP-2）.Ad-hBMP-2 was amplified by infection of 293A cells,and then the recombi- nant protein was analyzed by western blotting.Results:The target gene of hBMP-2 was transferred into Ad/CMV/V5-DEST vector cor- rectly with the right ORF（open reading frame）by LR recombination reaction and it was confirmed by restriction analysis,PCR and se- quencing.The expression clone ad-BMP-2 was packaged into maturated adenovirus successfully.The titer of Ad-hBMP-2 was 2.5×10⁹pfu/ml.It was confirmed with high-level expression of recombinant protein of hBMP-2 in 293A cells.Conclusions:It was the first time to construct Ad-hBMP-2 with an efficient and flexible system named Gateway^TMclone technology,and which lays a experimental foun-dation for the further research on physiological function of hBMP-2 and the genetic therapy of hBMP-2.更多还原