病毒载体介导β折叠阻断肽的表达及其对β淀粉样蛋白神经毒性的抑制作用

A viral vector expressing secretory β sheet breaker peptide inhibited Aβ neuronal toxicity

目的:通过病毒载体转染体外培养的海马神经元,观察后者表达分泌性的β折叠阻断肽(β-sheet breaker peptide,BSB)的生物活性。方法:采用分子克隆技术构建编码神经营养素4(neurotrophin-4,NT4)信号肽-BSB融合基因的重组腺伴随病毒(recombinant adeno-associated vrius,r AAV)载体,AAV在包装细胞系293细胞中进行包装,蔗糖梯度离心法纯化病毒颗粒,斑点杂交法测定重组病毒滴度。MTT检测及电镜观察BSB的生物学效应。结果:成功构建p SSHG/NT4-BSB质粒载体,纯化后获得滴度为3.6×1012~1.8×1013/m L的病毒颗粒。通过病毒感染培养的神经元分泌表达的BSB有效地抑制了β-淀粉样蛋白(Aβ)的纤维化聚集,明显降低了Aβ在体外的神经元毒性。结论:采用AAV载体介导的BSB短肽的分泌表达在体外培养的海马神经元中显示了良好的生物活性。

Objective: To observe biological activity of secretory β sheet breaker peptide( BSB) expressed via a viral vector in cultured hippocampal neurons. Methods: We constructed the recombinant adeno-associated virus( r AAV) encoding fusion gene of neurotrophin-4( NT-4) signal peptide and BSB by molecular cloning technique. The virus were produced in 293 packaging cell lines of the AAV and purified by sucrose gradient centrifugation. The viral titer was determined by dot blot hybridization. The biological effects of expressive BSB in vitro were observed by MTT assay and electron microscopy. Results: The p SSHG / NT4-BSB plasmid was constructed successfully. The physical titer of recombinant AAV was 1 × 1011-1 × 1012 virions / m L after purification. The BSB,secretive expression in AAV transfected cultured hippocampal neuron,inhibited Aβ fibrosis aggregation and significantly decreased the neurotoxicity of Aβ in cultured hippocampal neuron. Conclusion: AAV vector mediated secretive expression of BSB peptide displayed effective biological effect in vitro cultured neurons.