Construction and identification of recombinant adeno-associated virus vector encoding BDNF by Helper-Free system
Construction and identification of recombinant adeno-associated virus vector encoding BDNF by Helper-Free system
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发表时间：
2007
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通讯作者：
Chen Jia-feng
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作者：
Chen Jia-feng
目的：构建编码SD大鼠脑源性神经营养因子(BDNF)基因的腺相关病毒载体，并检测其体外表达BDNF基因的能力。方法：将BDNF特异性序列克隆到AAV-Free-Free系统中的pAAV-MCS中，构建BDNF表达载体pAAV-MCS-BDNF。经DNA测序和限制性内切酶酶切鉴定。用磷酸钙沉淀法将pAAV-MCS-BDNF与对照载体pAAV-RV、pHelper共转染包装细胞系HEK293细胞。用重组腺相关病毒载体感染HeLa细胞，用Western印迹、RT-PCR和ELISA法检测BDNF基因的表达。结果：成功构建了携带BDNF序列的重组腺相关病毒载体，病毒滴度为1.29×108，并能随时间的推移上调BDNF基因的表达。结论：重组腺相关病毒载体rAAV-BDNF能显著增强BDNF基因的表达，为其应用于神经损伤性疾病的治疗奠定了基础。
objective: To construct adeno-associated virus vector encoding the SD rat brain derived neurotrophic factor (BDNF) gene and examine its ability to express the BDNF gene in vitro. Methods: The specific BDNF sequence was cloned into the plasmid of pAAV-MCS in AAV helper-Free system to construct the BDNF expression plasmid pAAV-MCS-BDNF. The recombinant plasmids were identified by DNA sequencing and restriction digestion. Then the packaging cell lines (HEK293 cell) were cotransfected with the pAAV-MCS-BDNF together with the control plasmid pAAV-RV and pHelper by phosphate-calcium deposit method. The recombinant adeno-associated virus vector infected the hela cell, the expression of BDNF was detected by Western blot, RT-PCR and ELISA method, Results: The recombinant adeno-associated virus vector carrying the BDNF Sequence was constructed successfully, the viral titer was 1.29×108 and it could up-regulated the expression of the BDNF gene following with the time. Conclusions: The recombinant adeno-associated virus vector rAAV-BDNF can enhance the expression of BDNF gene significantly, which lays the basis for its application in the treatment of the neurodamaged disease.