重组人骨形态发生蛋白2腺病毒载体的构建与鉴定

Construction and identification of recombinant human bone morphogenetic protein-2 gene adenoviral vector

背景:目前报道的重组腺病毒构建方法较复杂,构建周期长,导致重组腺病毒构建的成功率较低。GatewayTM技术是基于λ噬菌体的位点特异性高效重组系统,其构建重组病毒载体具有快捷高效的特点。目的:构建含人骨形态发生蛋白2基因的重组腺病毒载体,为应用骨形态发生蛋白2基因治疗的研究奠定基础。设计、时间及地点:单一样本观察,实验于2008-02/06在深圳市第二人民医院中心实验室完成。材料:pOTB7-BMP-2质粒、HEK293为ATTC产品;BLOCK-iTTM Adenovirus Expression System为Invitrogen产品;大肠杆菌DH5α为Biontex产品。方法:用聚合酶链反应方法扩增骨形态发生蛋白2目的基因,并插入载体pMD19-T Simple Vector中进行测序分析。将骨形态发生蛋白2基因亚克隆到穿梭载体pEC3.1(+)中,构建pEC3.1-BMP-2穿梭质粒,再将其中的表达盒通过重组克隆到pAd/BLOCK-iT?-DEST腺病毒载体中,线性化后转293细胞进行包装获得重组腺病毒rAd-BMP2。主要观察指标:①目的基因骨形态发生蛋白2的聚合酶链反应扩增。②重组质粒TS-BMP2的构建。③重组pEC3.1-BMP2的构建。④重组pAd-BMP2的构建。⑤重组腺病毒的制备与鉴定。结果:正确扩增长约1.2kb的骨形态发生蛋白2cDNA,并成功地构建其腺病毒载体,经线性化的pAd-BMP2DNA转染HEK293细胞,包装、扩增后得到人骨形态发生蛋白2重组腺病毒(rAd-BMP2),其滴度约为1×1010PFU/mL,该滴度可满足进一步的骨形态发生蛋白2成骨作用的研究。结论:成功地构建重组人骨形态发生蛋白2腺病毒载体。

BACKGROUND： Previous studies regarding recombinant adenovirus is characterized by complicated construction method with long period, which lead to low success rate. By using site-specific recombination, GatewayTM technique can construct recombinant virus vector effectively and rapidly. OBJECTIVE： To construct recombinant adenovirus vector carrying human bone morphogenetic protein-2 （hBMP-2）, in order to provide a basis for the research of gene therapy. DESIGN, TIME AND SETTING： A single sample observation was performed at the Central Laboratory of Shenzhen Second People＇s Hospital between February and June 2008. MATERIALS： pOTB7-BMP-2 plasmid, HEK293 was produced by ATTC. BLOCK-iTTM Adenovirus Expression System was purchased from Invitrogen Company. E. coil DH5α was supplied by Biontex. METHODS： BMP-2cDNA was amplified by PCR, and then inserted into cloning vector pMD19-T Simple Vector. The BMP-2cDNA was subcloned into a shuttle vector and the recombinant pEC3.1-BMP2 vector was obtained. It was subcloned into pAd/BLOCK-iT^TM-DEST adenoviral genomic and linearization transfection 293 cell package to obtain Ad-BMP2. MAIN OUTCOME MEASURES： ①Amplification of BMP-2 by PCR. -②Construction of recombinant plasmid TS-BMP2, pEC3.1-BMP2 and pAd-BMP2. ③Preparation and identification of recombinant adenovirus. RESULTS： A length of 1 200 bp BMP-2 cDNA was amplified, which was subcloned into adenoviral vector, linearizing pAd-BMP2 DNA followed by transfected HEK293 cells and the rAd-BMP2 was obtained. The titre of virus was 1 × 10^10 PFU/mL, which could meet the need of research of BMP2 osteogenesis. CONCLUSION： The hBMP-2 recombinant adenovirus is constructed successfully.