采用Gateway^TM技术构建人骨形态发生蛋白-2基因重组腺病毒载体

目的：采用基于attLXattR的入噬茵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2.5×10^9pfu／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入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。

Construction of Recombinant Adenovirus Vector for hBMP-2 Gene with Gateway^TM Clone Technology

Objective： To establish a recombinant adenovirus vector with BMP-2 by λ phasge-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 Ⅰ, 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 recombinant protein was analyzed by western blotting. Results： The target gene of hBMP-2 was transferred into Ad/CMV/V5-DEST vector correctly with the right ORF （open reading frame） by LR recombination reaction and it was confirmed by restriction analysis, PCR and sequencing. The expression clone ad-BMP-2 was packaged into maturated adenovirus successfully. The titer of Ad-hBMP-2 was 2.5 × 10^9pfu/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 GatewayTM clone 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.