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目的为了研究蝎毒癫痫肽对癫痫模型的治疗作用,构建并产生重组腺伴病毒(AAV)载体。方法将AEP外源性核酸片段插入AAV-shufer质粒pSSHG-Neo的KpnI-BamH I位点构建AAV。用腺病毒辅助质粒pFG140、包装质粒pAAV/Ad及构建的pSSHG-AEP三质粒磷酸钙共沉淀法在肾胚胎293细胞系中同源重组包装rAAV。使用地高辛标记的斑点杂交方法测定重组病毒滴度。结果成功构建了重组病毒质粒pSSHG/AEP,经蔗糖梯度离心法获得rAAV组分,梯度稀释测得滴度为1.46×1012PFU/mL。结论成功地制备了AEP重组腺伴随病毒(rAAV/AEP)载体,为癫痫基因治疗实验奠定了基础。
Objective To study the therapeutic effect of epilepsy peptide from scorpion venom on epilepsy model and construct recombinant adeno-associated virus (AAV) vector. Methods AAV was constructed by inserting AEP exogenous nucleic acid into KpnI-BamH I site of AAV-shufer plasmid pSSHG-Neo. RAAV was packaged by homologous recombination in the kidney embryonic 293 cell line using the adenovirus helper plasmid pFG140, the packaging plasmid pAAV / Ad and the constructed pSSHG-AEP tri-plasmid calcium phosphate co-precipitation method. Recombinant virus titers were determined using dot blot hybridization with digoxigenin. Results The recombinant virus plasmid pSSHG / AEP was successfully constructed. The rAAV fraction was obtained by sucrose gradient centrifugation. The titer was 1.46 × 1012 PFU / mL. Conclusion AEP recombinant adeno-associated virus (rAAV / AEP) vector was successfully prepared, which laid the foundation for the gene therapy experiment of epilepsy.