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目的:构建人骨保护素基因的重组腺病毒载体,并观察在大鼠骨髓基质细胞中的转染能力及表达效率。方法:实验于2004-03/2005-04在第三军医大学完成。采用基因工程技术,将人骨保护素基因片段插入到腺病毒载体质粒pAdTrack-巨细胞病毒上,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,得到携带人骨保护素基因的重组腺病毒,将携带人骨保护素基因重组腺病毒对大鼠骨髓基质细胞进行感染。采用聚合酶链反应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,应用Westernblot及酶联免疫吸附法检测骨保护素在大鼠骨髓基质细胞中的表达。结果:①酶切鉴定及聚合酶链反应结果证明人骨保护素基因重组腺病毒载体构建成功,病毒滴度可达2.5×109pfu/mL,具有很强感染能力。②人骨保护素基因重组腺病毒载体转染大鼠骨髓基质细胞后48h,Westernblot及酶联免疫吸附法可检测到人骨保护素的蛋白表达。结论:应用细菌内同源重组法,可成功构建含人骨保护素基因重组腺病毒载体,且能高效转染大鼠骨髓基质细胞。
OBJECTIVE: To construct the recombinant adenovirus vector of human osteoprotegerin gene and observe its transfection efficiency and expression efficiency in rat bone marrow stromal cells. Methods: The experiment was performed at the Third Military Medical University from March 2004 to April 2005. Using gene engineering technique, the human osteoprotegerin gene fragment was inserted into the adenoviral vector plasmid pAdTrack-cytomegalovirus, homologous recombination was carried out in the intracellular of E. coli BJ5183 using the PadEasy system, and the recombinant plasmid was packaged and expanded in 293 cells to obtain Recombinant adenovirus carrying human osteoprotegerin gene will infect rat bone marrow stromal cells infected with recombinant adenovirus carrying human osteoprotegerin gene. The recombinant adenovirus vector was identified by polymerase chain reaction (PCR). The green fluorescent reporter gene transfection results were used to detect the titer of recombinant adenovirus by fluorescence counting. Western blotting and enzyme-linked immunosorbent assay were used to detect the expression of osteoprotegerin in rat Bone marrow stromal cells. Results: (1) The results of restriction enzyme digestion and polymerase chain reaction proved that the human osteoprotegerin gene recombinant adenovirus vector was successfully constructed with a titer of 2.5 × 109pfu / mL and strong infectivity. (2) Forty-eight hours after transfection of human osteoprotegerin gene recombinant adenovirus vector into rat bone marrow stromal cells, the expression of human osteoprotegerin was detected by Western blot and enzyme-linked immunosorbent assay. CONCLUSION: Recombinant adenoviral vector containing human osteoprotegerin gene can be successfully constructed by homologous recombination in bacteria and can efficiently transfect rat bone marrow stromal cells.