pAAV-hBMP2-IRES质粒的构建及病毒包装  

Construction and package of pAAV-hBMP2-IRES plasmid

作　　者：程政[1] 王敏[1] 石建峰[1] 时志斌[2] 

机构地区：[1]西安交通大学口腔医院,陕西西安710004 [2]西安交通大学第二附属医院

出　　处：《临床口腔医学杂志》2009年第1期13-15,共3页Journal of Clinical Stomatology

基　　金：陕西省卫生厅科学研究基金(06D40);陕西省科技计划项目(2006K14-G3);西安交通大学口腔医院青年基金(0603)

摘　　要：目的:构建含人骨形态发生蛋白2(hBMP2)基因的重组腺相关病毒载体。方法:根据hBMP-2基因序列设计引物,上游引入SalI酶切位点,下游引入EcoRI酶切位点,以胚胎骨髓组织总RNA为模版进行rt-PCR反应扩增hBMP-2基因。将hBMP-2片段定向亚克隆进pAAV-IRES-GFP的MCS中,获得重组表达质粒(pAAV-hBMP2-IRES-GFP),采用磷酸钙沉淀法将该质粒与包装质粒pAAV-RC和辅助质粒pHelper共同转染到AAV293细胞,进行rAAV-hBMP2-IRES-GFP重组病毒包装。收获的病毒液按氯仿处理、PEG/NaCl沉淀、氯仿反复抽提法浓缩纯化,采用荧光计数法测定重组病毒感染滴度。结果:酶切鉴定、测序结果表明,hBMP2成功克隆入pAAV-IRES-GFP载体中。在AAV293细胞中包装出重组腺相关病毒载体的感染滴度为3.6×1011/ml。结论:成功制备了rAAV-hBMP2载体,可满足骨组织工程的需要。Objective： To construct the recombinant adeno-associated virus vector with human bone morphonenetic protein 2 gene （rAAV-hBMP2）. Method： The moulds which contained hBMP-2 vector were amplified by PCR. The reclaimed productions and pAAV-IRES-GFP were digested and integrated,bacillus coli DH5α competent cells were trans- formed,and recombinant plasmid pAAV-hBMP2-IRES-GFP was obtained. 293 cells were virus-encloded by the calcium phosphate precipitation method and the virus fluid was obtained and processed by chloroform precipitate treatment, PEG8000 / NaCI precipitation and chloroform extraction for purification. Virus titer was identified by fluorescence counting method. Result： Calcium phosphate coprecipitation methods were used in 293 cells and adeno-associated viral vector rAAV-IRES-hBMP2 was recombinated. The virus titer was about 3.6×10^11 / mh Conclusion： With this method, rAAV- hBMP2 can be acquired successfully and it is useful to bone tissue engineering.

关 键 词：人骨形态发生蛋白 腺相关病毒 骨组织工程 

分 类 号：R554.1[医药卫生—血液循环系统疾病] R378.99[医药卫生—内科学]