人骨保护素基因重组腺病毒载体在大鼠骨髓基质细胞中的表达效率  

Expressive efficiency of recombinant adenovirus vector of human osteoprotegerin gene in bone marrow stroma cell in rats

作　　者：王晓军[1] 王爱民[1] 张忠荣[1] 吴思宇[1] 郭庆山[1] 贺伟峰[2] 汤守营[3] 张全顺[3] 

机构地区：[1]解放军第三军医大学大坪医院野战外科研究所全军战创中心骨创伤科,重庆市400042 [2]解放军第三军医大学西南医院烧伤研究所,重庆市400038 [3]解放军第二五一医院骨科,河北省张家口市075000

出　　处：《中国临床康复》2005年第34期4-7,i0001,共5页Chinese Journal of Clinical Rehabilitation

摘　　要：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。AIM：To construct the recombinant adenovirus vector of human osteoprotegerin （hOPG）gene and to observe the transfection ability and expressive efficiency in bone marrow stroma cells in rats. METHODS： The experiment was conducted at the Third Military Medical University from March 2004 to April 2005. By the method of DNA recombination technique, the fragment of hOPG gene encoding region was cloned into the shuttle plasmid pAdTrack-cytomegalovirus （CMV）, with the PadEasy system and the backbone plasmid, the homologous recombination took place in the escherichia coli BJ5183. The recombinant adenovirus that took the hOPG was generated after the package and amplification of the 293 cells. The bone marrow stroma cells in the rats were infected with the recombinant adenovirus that took along the hOPG. The recombinant adenovirus vector was evaluated with the polymerase chain reaction （PCR）. The titre of recombinant adenovirus was detected with fluorescent counting method. The expression of hOPG in bone marrow stroma cells of rats was assessed with Western blot and enzyme-linked immuno-sorbent assay （ELISA）. RESULTS： ① Restriction endonuclease and PCR analyses confirmed that the hOPG gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus could reach 2.5×10^9 pfu/mL. The adenovirus had a strong effective ability. ② The expression of hOPG was determined by Western blot and ELISA in bone marrow stroma cells after 48 hours transfection. CONCLUSION： By the method of homogenous recombination in bacteria, the recombinant adenovirus containing hOPG gene can be successfully constructed and can be transferred into bone marrow stroma cells effectively in rats.

关 键 词：腺病毒科 基因疗法 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]