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目的构建能够表达神经生长因子β(nerve growth factorβ,NGF-β)的重组腺相关病毒(adenoassociatedvirus,AAV)穿梭质粒,为进一步包装AAV奠定基础。方法利用高保真酶分别扩增pAAV-多克隆位点(multiple cloning site,MCS)的结构元件和pGenesil-1.1的功能元件,连入T-easy载体,限制性核酸内切酶酶切并回收目的片段,DNA连接酶连接,得到含U6和CMV双启动子的重组AAV穿梭质粒pAAV-U6/CMV-增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP),用该质粒转染293细胞,观察有无绿色荧光表达;培养人Miapaca-2细胞,提取总RNA,进行RT-PCR得到NGF-β基因,插入T-easy载体,得到T-easy-NGF-β;将NGF-β插入pAAV-U6/CMV-EGFP,构建NGF-β重组AAV穿梭质粒pAAV-U6/CMV-NGF-β,将该质粒送基因测序。结果双酶切pAAV-U6/CMV-EGFP可见4 250、800 bp条带,质粒转染293细胞后可见绿色荧光表达;质粒T-easy-NGF-β双酶切可见3 015、736 bp条带,测序显示基因序列正确;双酶切pAAV-U6/CMV-NGF-β可见4 250、736 bp条带;质粒pAAV-U6/CMV-NGF-β测序显示载体中的基因序列正确。结论成功构建NGF-β重组AAV穿梭质粒,为进一步深入研究脊髓损伤的基因治疗提供了实验基础。
Objective To construct recombinant adenovirus-associated virus (AAV) shuttle plasmid capable of expressing nerve growth factor β (NGF-β), and lay the foundation for further packaging of AAV. Methods High-fidelity enzymes were used to amplify the structural elements of pAAV-multiple cloning site (MCS) and functional elements of pGenesil-1.1, respectively, and ligated into the T-easy vector. Restriction endonucleases were digested and recovered Fragment and DNA ligase to obtain the recombinant AAV shuttle plasmid pAAV-U6 / CMV-enhanced green fluorescent protein (EGFP) containing the U6 and CMV double promoters. The 293 cells were transfected with the plasmid, The expression of NGF-β was detected by RT-PCR and inserted into the T-easy vector to obtain T-easy-NGF-β. NGF-β was inserted into pAAV-U6 / CMV-EGFP to construct NGF-β recombinant AAV shuttle plasmid pAAV-U6 / CMV-NGF-β, the plasmid was sent gene sequencing. Results The double digested pAAV-U6 / CMV-EGFP showed a band of 4 250,800 bp. The plasmid was transfected into 293 cells and the green fluorescent protein was detected. The 3 015 and 736 bp bands were digested with T-easy-NGF-β , And the gene sequence was confirmed by sequencing. The 4 250,736 bp band was digested with pAAV-U6 / CMV-NGF-β by double enzyme digestion, and the sequence of plasmid pAAV-U6 / CMV-NGF-β was correct. Conclusion The successful construction of NGF-β recombinant AAV shuttle plasmid provides an experimental basis for further study of gene therapy of spinal cord injury.