论文部分内容阅读
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 a recombinant adeno-associated virus vector containing human bone morphogenetic protein 2 (hBMP2) gene. Methods: Primers were designed according to hBMP-2 gene sequence. The upstream site of SalI was introduced into SalI restriction site and the downstream of EcoRI restriction site. The hBMP-2 gene was amplified by rt-PCR from total RNA of embryonic bone marrow. The hBMP-2 fragment was subcloned into the MCS of pAAV-IRES-GFP to obtain the recombinant expression plasmid (pAAV-hBMP2-IRES-GFP). The plasmid was cloned by calcium phosphate precipitation into the plasmid pAAV-RC and the pHelper Were co-transfected into AAV293 cells for rAAV-hBMP2-IRES-GFP recombinant virus packaging. Harvest the virus solution by chloroform treatment, PEG / NaCl precipitation, chloroform extraction by repeated extraction and purification, determination of recombinant virus titer by fluorescence counting. Results: The results of restriction enzyme digestion and sequencing showed that hBMP2 was successfully cloned into pAAV-IRES-GFP vector. The infectious titer of the recombinant adeno-associated virus vector packaged in AAV293 cells was 3.6 × 10 11 / ml. Conclusion: The rAAV-hBMP2 vector was successfully prepared to meet the needs of bone tissue engineering.