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Construction and preparation of recombinant adeno-associated virus vector for expressing shRNA targeting VEGF165 mRNA
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 22
- DOI:
- 10.3321/j.issn:1000-2790.2007.22.003
- Key Word:
- 血管内皮生长因子类;腺相关病毒;遗传载体;RNA干扰;短发卡样RNA
Abstract： 目的:构建并制备携带靶向干扰VEGF165基因shRNA的重组腺相关病毒载体.方法:将PCR法扩增所得EGFP-U6-shRNA(VEGF165)片段插入载体质粒pSNAV2.0-lacz-α的EcoRI和SalI酶切位点,构建重组质粒pSNAV2.0-EGFP-U6-shRNA(VEGF165).以HSV1-RapCap/△UL2为辅毒,包装rAAV2-EGFP-U6-shRNA(VEGF165)病毒.对所获病毒载体进行PCR,SDS-PAGE鉴定分析、滴度测定和EGFP的活性检测.结果:PCR,SDS-PAGE鉴定分析结果表明靶向干扰VEGF165基因的shRNA片段成功包装入重组AAV2载体,病毒纯度在98%以上.点杂交法测定重组病毒载体基因组滴度约为3×1014 v.g.(vector genomes)/L. EGFP活性检测显示重组AAV2感染细胞阳性率在30%以上.结论:成功制备了rAAV2-EGFP-U6-shRNA(VEGF165)病毒载体,为通过RNA干扰技术特异性抑制VEGF基因的表达来阐明VEGF在缺血性脑损伤后各种生物学事件中的作用奠定了实验基础.
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