BDNF基因重组逆转录病毒表达载体pLEGFP-BDNF的构建与鉴定

Construction and identification of recombinant retroviral vector carrying rat BDNF gene

目的构建脑源性神经营养因子(BDNF)基因重组逆转录病毒表达载体。方法根据 BDNF基因已知序列,设计合成一对引物并导入HindⅢ和BamH Ⅰ酶切位点;从大鼠海马组织提取总 RNA,逆转录聚合酶链反应(RT-PCR)获得编码BDNF的基因片段,与克隆载体pMD 18-T Simple连接构建pMDT-BDNF质粒;经HindⅢ、BamHⅠ双酶切,获得BDNF基因片断再克隆至逆转录病毒载体 pLEGFP-N1中构建重组质粒pLEGFP-BDNF。结果限制性内切酶酶切分析和PCR法鉴定表明为正确重组子,测序结果证实与已知序列吻合。结论构建的重组逆转录病毒表达载体 pLEGFP-BDNF含有序列正确的大鼠BDNF基因,可以作为今后治疗老年性痴呆动物模型转基因实验的基因来源。

Objective To construct recombinant retroviral vector carrying rat BDNF gene. Methods Total RNA was extracted from rat brain hippocampus as the template and the BDNF gene was amplified by reverse transcription-polymerase chain reaction （RT-PCR） with a pair of specified primers containing the restriction sites of BamH Ⅰ and Hind Ⅲ. The amplified fragment of BDNF gene was cloned into cloning vector pMD 18-T Simple, digested with BamH Ⅰ and Hind Ⅲ and then subcloned into the expression vector pLEGFP-NI. Results The recombinant plasmid was identified by restriction endonuclease analysis and PCR and DNA sequencing. Conclusion The recombinant retroviral vector carrying rat BDNF gene can be constructed successfully, which offers a help for the further research on gene therapy of Alzheimer disease.