表达大鼠脑源性神经营养因子(BDNF)腺相关病毒的构建及体外表达分析

Construction and identification of recombinant adeno-associated virus vector encoding BDNF by Helper-Free system

目的构建携带大鼠脑源性神经营养因子(BDNF)基因的重组腺相关病毒(AAV)载体,并检测体外表达目的基因的能力。方法应用基因克隆技术将大鼠BDNF的cDNA基因序列克隆入腺病毒质粒pAAV-MCS,PCR酶切测序鉴定序列。与AAV病毒包装质粒pAAV-RC、pHelper用磷酸钙法共转染HEK293细胞,包装得到含转基因过表达BDNF的病毒载体(rAAV-BDNF)。重组病毒感染体外培养的Hela细胞后,用RT-PCR和Western Blot方法检测细胞BDNF基因及蛋白表达情况,用ELISA法测定培养液中表达产物浓度。结果证实BDNFcDNA片段插入到病毒基因组内,并整合到宿主基因组后稳定表达,病毒滴度可以达到1.29×108PFU/mL。重组病毒感染Hela细胞后能高水平表达BDNF,ELISA结果表明,Hela细胞从病毒感染后第2天到第10天表达BDNF水平呈上升趋势,第10天达到2253pg/mL。结论成功地构建了表达BDNF基因的腺相关病毒载体,并可在体外高水平表达其所携带的目的基因,为将其进一步应用于神经系统损伤性疾病治疗的研究奠定了方法学基础。

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-BDIqF 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 × 10^8 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.