新型表达骨形态发生蛋白2腺病毒真核细胞载体的构建和鉴定.doc

　　新型表达骨形态发生蛋白2腺病毒真核细胞载体的构建和鉴定 ：张正，刘丹平，陈峻江，郭韬，张男 【摘要】 目的 构建同时表达具有抗原表位FLAG标记的骨形态发生蛋白2(BMP2)目的蛋白和报告分子绿色荧光蛋白(GFP)的新型腺病毒真核细胞表达载体。方法 将去除翻译终止密码子并添加新的酶切识别位点XhoⅠ的BMP2基因定向连入腺病毒穿梭质粒pShuttle CMV-IRES-hrGFP-1。携带BMP2+基因的重组腺病毒穿梭质粒经酶切鉴定、PmeⅠ酶切线性化后转化BJ5183-AD-1大肠杆菌，利用细菌内同源重组机制将BMP2+和GFP基因连同其顺势表达元件重组入腺病毒基因组质粒。用PacⅠ酶切去除其质粒成分，暴露腺病毒基因组的反向末端重复序列，转染293细胞，进行腺病毒的包装，完成新型腺病毒载体Ad CMV-BMP2+-IRES-GFP-1的构建。作为阳性对照，同时制备多年来广为应用的 BMP2腺病毒表达载体Ad CMV-BMP2。以两种载体感染骨髓基质细胞，通过RT-PCR和荧光显微镜分别检测骨形态发生蛋白2和绿色荧光蛋白表达情况;通过碱性磷酸酶活性检测判断BMP2诱导成骨分化作用 。结果 突变后骨形态发生蛋白2基因序列、重组腺病毒穿梭质粒和重组腺病毒质粒酶切图谱符合预定设计。感染骨髓基质细胞后，新型腺病毒载体可同时表达报告分子GFP和具有诱导成骨分化活性的BMP2。结论 成功构建表达BMP2的新型腺病毒载体。 【关键词】 骨形态发生蛋白2基因;绿色荧光蛋白;腺病毒载体;同源重组;成骨诱导 Abstract: Objective To construct a novel rebinant adenovirus vector expressing the BMP2 fused to FLAG epitope and green fluorescent protein(GFP) as a reporter on the same transcript. Methods The basic pairs of BMP2 behind the translation stopping codon TAG oved and a XhoⅠrestriction site utant. Then, the mutant of BMP2 gene (BMP2+ gene)ultiple cloning sites of the adenovirus shuttle plasmid pShuttle CMV-IRES-hrGFP-1 by means of the directional cloning technology. Once tested by restriction digestion, the shuttle plasmid e I and transformed into BJ5183-AD-1 cells to rebine the genes of BMP2+ and GFP together ents into adenoviral plasmid through a neplified bacterial homologous rebinant method. To expose its inversted terminal repeats, the rebinant adenoviral plasmids arroal cells (MSCs), the expression of BMP-2 gene ed by alkaline phosphatase activity assay, and the expression of GFP gene icroscope. As a positive contrast, another rebinant adenovirus, AdCMV-BMP2, any years ago, utant or the restriction maps of the rebinant adenoviral shuttle plasmids and the rebinant adenoviral plasmids accorded ental project beforehand. The nee time. The expressed BMP2 had osteoinduction activity. Conclusions A novel rebinant adenovirus vector expressing the BMP2 fused to FLAG epitope and green fluorescent protein as a reporter on