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Construction of recombinant adenoviral plasmid bearing hSDF-1α cDNA by homologous recombination in bacteria and preparation of recombinant adenovirus expressing hSDF-1α
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 5
- DOI:
- 10.3321/j.issn:1000-2790.2007.05.001
- Key Word:
- 同源重组;人基质细胞源衍生因子-1;腺病毒;基因治疗
Abstract： 目的:利用细菌内同源重组快速构建携带hSDF-1α cDNA重组腺病毒质粒和制备表达hSDF-1α重组腺病毒. 方法: 制备感受态BJ5183,将pAdEasy-1转化BJ5183,制备含pAdEasy-1的BJ5183感受态,将线性化的pShuttle-EGFP-hSDF-1α转化含pAdEasy-1的BJ5183感受态菌. 采用细菌中同源重组法构建重组腺病毒质粒pAd-EGFP-hSDF-1α. pAd-EGFP-hSDF-1α用Pac Ⅰ线性化后,再用LipoVec介导其转染至AD293细胞内包装扩增出重组腺病毒颗粒,采用CsCl密度梯度离心法纯化重组腺病毒Ad-EGFP-hSDF-1α. 采用荧光显微镜下观察LipoVec介导的重组腺病毒质粒的转染效果及病毒包装情况. 结果:pShuttle-EGFP-hSDF-1α成功地转化了含pAdEasy-1的BJ5183,并在其内发生了同源重组. 荧光显微镜下观察证实了pAd-EGFP-hSDF-1α转染AD293后,产生了重组腺病毒Ad-EGFP-hSDF-1α. 病毒滴度为4.1×1015 pfu/L. 结论:用细菌内同源重组法可快速、高效制备表达hSDF-1α的高滴度重组腺病毒. 为研究hSDF-1α在动员骨髓源干细胞迁移到组织损伤部位修复组织损伤的研究奠定了基础.
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