pcDNA3/BDNF真核表达载体的构建

背景：脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)作用广泛，但属于生物大分子，不能通过血脑屏障。基因治疗是目前解决脑源性神经营养因子给药途径最有希望的方案。目的：拟构建大鼠脑源性神经营养因子基因真核表达载体。方法：采用反转录聚合酶链式反应技术从SD大鼠脑组织提取总RNA，扩增脑源性神经营养因子基因cDNA序列，并将其克隆到真核表达载体pcDNA3中，分别取10 g质粒pcDNA3和纯化的目的基因分别进行EcoR Ⅰ、xho Ⅰ双酶切。将目的基因片段和pcDNA3载体连接，转入感受态DH5α细胞中，经酶切鉴定后送上海博亚生物技术有限公司测序。结果与结论：RT-PCR产物为749 bp的特异片段，重组质粒pcDNA3/BDNF酶切后产生 749 bp和5 446 bp的片段，DNA测序证实749 bp片段的碱基序列与大鼠脑源性神经营养因子基因序列完全一致，成功构建了pcDNA3/BDNF重组质粒。

Construction of pcDNA3/BDNF eukaryotic expression vectors

BACKGROUND:Brain-derived neurotrophic factor (BDNF) is biomacromolecule, which can not pass through the blood-brain barrier. Now gene therapy is the most promising program to solve route of administrationOBJECTIVE:To construct the eukaryotic expression recombinant plasmid pcDNA3/BDNF. METHODS:Total RNA was extracted from Sprague Dawley rats using RT-PCR. By gene recombination technique, rat BDNF coding sequence was inserted into eukaryotic expression vector pcDNA3. The fragment of target gene was connected with pcDNA3 vector and transfected into DH5α cells, and recombinant plasmid was verified with restriction enzyme digestion and DNA sequencing.RESULTS AND CONCLUSION:RT-PCR showed that BDNF product was 749 bp specific segment. By restriction enzyme digestion, the recombinant plasmid consisted to 749 bp and 5 446 bp fragments. The DNA sequence of the 749 bp fragment was identical with rat BDNF cDNA in Gene Bank. The recombinant plasmid pcDNA3/BDNF was constructed successfully.