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目的构建人骨保护素(hOPG)基因的重组腺病毒载体并检测其转染能力。方法采用基因工程技术,将hOPG基因片段插入到腺病毒载体质粒pAdTrack-CMV上,利用PadEasy系统与骨架质粒在大肠杆菌BJ5183胞内进行同源重组,经293细胞包装、扩增,得到携带hOPG基因的重组腺病毒AdEasy-PAdtrack-CMV-hOPG,将重组腺病毒AdhOPG对大鼠骨髓基质细胞(rMSCs) 进行感染。采用PCR方法对重组腺病毒载体进行鉴定,利用绿色荧光报告基因转染结果,以荧光计数法检测重组腺病毒的滴度。结果酶切鉴定及PCR结果证明hOPG基因重组腺病毒载体成功, 病毒滴度可达2．5×108pfu／ml,具有很强感染能力,结论应用细菌内同源重组法,成功构建了含 hOPG重组腺病毒载体。
Objective To construct the recombinant adenovirus vector of human osteoprotegerin (hOPG) gene and test its transfection ability. Methods The hOPG gene fragment was inserted into the adenoviral plasmid pAdTrack-CMV by genetic engineering and homologous recombined with the backbone plasmid in E. coli BJ5183 using PadEasy system. The hOPG gene was packaged and amplified by 293 cells, and the hOPG gene Of recombinant adenovirus AdEasy-PAdtrack-CMV-hOPG, the recombinant adenovirus AdhOPG on rat bone marrow stromal cells (rMSCs) were infected. The recombinant adenovirus vector was identified by PCR. The green fluorescent reporter gene transfection results were used to detect the titer of recombinant adenovirus by fluorescence counting. Results The restriction endonuclease digestion and PCR results proved that the hOPG gene recombinant adenovirus vector was successful and the virus titer was up to 2.5 × 108pfu / ml, which indicated that the hOPG gene had a strong ability to infect. CONCLUSIONS: Adenovirus vector.