【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。更多还原

【Abstract】 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 re-claimed 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 3.6×1011/ml.Conclusion:With this method,rAAV-hBMP2 can be acquired successfully and it is useful to bone tissue engineering.更多还原