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背景:人血管内皮生长因子121和骨形态蛋白2在激素性股骨头坏死骨缺损中均具有成血管成骨作用,目前国内在以人血管内皮生长因子121和骨形态蛋白2基因联合治疗激素性股骨头坏死方面少有报道。目的:构建人血管内皮生长因子121与人骨形态发生蛋白2双基因腺病毒穿梭质粒。方法:将质粒pShuttle-CMV-VEGF121-IRES-hrGFP-1经KpnⅠ/XbaⅠ酶切后,将BMP2片段定向连入pShuttle-CMV-VEGF121-IRES,构建可同时表达2个目的基因重组质粒pShuttle-CMV-VEGF121-IRES-BMP2,注入H5a细胞扩增,铺板,筛选阳性菌落,提取质粒,进行酶切分析及序列测定。将已构建确认正确的腺病毒质粒,经BJ5183-AD-1电转感受态细胞进行电穿孔后,铺板,筛选阳性菌落,提取质粒,进行酶切分析,PCR检测和序列分析。结果与结论:酶切分析及核酸序列测定证实重组质粒构建正确,提示实验成功构建了血管内皮生长121及骨形态蛋白2双基因共表达重组腺病毒载体。
BACKGROUND: Human vascular endothelial growth factor 121 and bone morphogenetic protein 2 play an important role in osteogenesis in steroid-induced osteonecrosis of the femoral head. At present, the combination of human vascular endothelial growth factor 121 and bone morphogenetic protein 2 Few reports of osteonecrosis. OBJECTIVE: To construct human adenovirus shuttle plasmid of human vascular endothelial growth factor 121 and human bone morphogenetic protein 2. Methods: The recombinant plasmid pShuttle-CMV-VEGF121-IRES-hrGFP-1 was digested with KpnⅠ / XbaⅠand inserted into pShuttle-CMV-VEGF121-IRES. -VEGF121-IRES-BMP2. H5a cells were injected into H5a cells for amplification, plating and screening of positive colonies. Plasmids were extracted and analyzed by restriction enzyme digestion and sequencing. After confirming the correct construction of the adenovirus plasmid, the competent cells were electroporated with BJ5183-AD-1 electroporation. The positive colonies were screened out and positive plasmids were extracted. The plasmids were extracted and subjected to restriction analysis, PCR detection and sequence analysis. RESULTS AND CONCLUSION: The results of enzyme digestion analysis and DNA sequencing confirmed that the recombinant plasmids were correctly constructed, which indicated that recombinant adenovirus vector coexpressing vascular endothelial growth factor 121 and bone morphogenetic protein 2 gene was constructed successfully.