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人神经生长因子重组腺伴随病毒载体的构建 被引量：1

Construction of the vector of human nerve growth factor recombinant adeno-associated virus
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摘要 目的　为了研究人神经生长因子 (hNGF)基因导入急性脊髓损伤 (SCI)模型的治疗作用 ,构建并产生重组腺伴随病毒 (AAV)载体。方法　将hNGF外源性核酸片段插入AAV shuffer质粒 pSSHG Neo的KpnⅠ BamHⅠ位点构建AAV。用腺病毒辅助质粒 pFG14 0代替野生型腺病毒 ,包装质粒 pAAV/Ad及已构建的pSSHG Neo三质粒磷酸钙共沉淀法在肾胚胎 2 93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/hNGF ,经蔗糖梯度离心法获得rAAV组分 ,梯度稀释测得滴度为 1.4 6× 10 12 PFU·mL-1。结论　成功地制备了人神经生长因子重组腺伴随病毒 (rAAV/hNGF)载体 ,为SCI基因治疗实验奠定了基础。 Objective To investigate the treatment of spinal cord injury (SCI) by recombinant adeno-associated virus (AAV) transducing the hNGF gene, and construct and produce the vector of hNGF recombinant AAV. Methods The resulting gene of hNGF was inserted into the KpnⅠ-BamHⅠ site of vector plasmid pSSHG-Neo to construct the vector of hNGF recombinant AAV. The recombinant AAV viral stock was packaged. Renal embryo 293 cell was co-transfected with the rAAV vector of plasmid pSSHG/hNGF, packaging plasmid pAAV/Ad and helper adenovirus pasmid pFG140 instead of adenovirus by calcium phosphate precipitation. Results The recombinant viral stock vector of plasmid pSSHG/hNGF was constructed successfully. The results of dot blot showed that we had obtained the rAAV stocks of high titre 1.46×10 12 PFU·mL -1. Conclusion We prepared the viral stock of rAAV-hNGF that can serve as the experimental study of gene therapy of SCI.
作者 马巍 刘淼 杨广笑 王全颖
机构地区 西安交通大学第一医院骨科 西安华广生物工程有限公司
出处 《西安交通大学学报（医学版）》 CAS CSCD 北大核心 2004年第3期226-228,共3页 Journal of Xi’an Jiaotong University（Medical Sciences）
关键词 hNGF基因 重组腺伴随病毒(rAAV) 基因转移 human nerve growth factor gene recombinant adeno-associated virus gene transfer
分类号 R749.1 [医药卫生—神经病学与精神病学]
  • 相关文献

参考文献8

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二级参考文献8

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共引文献7

同被引文献18

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西安交通大学学报（医学版）
2004年 第3期

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