携带靶向干扰VEGF基因shRNA重组腺相关病毒载体的构建和制备

Construction and preparation of recombinant adeno-associated virus vector for expressing shRNA targeting VEGF165 mRNA

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/ΔUL2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3×1014v.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.

AIM： To construct and prepare the recombinant adeno-associated virus （AAV） vector for expressing short hairpin RNA （ shRNA ） targeting VEGF165 mRNA. METHODS ： By inserting the sequence that contained EGFP-U6- shRNA （targeting VEGF165 mRNA） into the EcoRI and SalI site of vector plasmid pSNAV2.0-lacz-α, we constructed the recombinant vector of plasmid pSNAV2.0- EGFP-U6- shRNA （ VEGF165 ）. The recombinant vector of plasmid, BHK cell lines and helper virus HSV1-RapCap/△UL2 were used for the package of the serotype 2 of recombinant adeno-associated virus（rAAV2） vector. The identity, viral purity, titer, EGFP activity of rAAV2 viral stock were analyzed by the methods of PCR, SDS-PAGE, dot-blot, and virus infection, respectively. RESULTS ： The results of PCR and SDSPAGE indicated that the shRNA fragment targeting VEGF165 was sucessfully packaged into rAAV2, and the viral purity of rAAV2 was above 98%. The titer of rAAV2 was approximately 3 × 10^14 v.g. （ vector genomes）/L, and the positive rate of rAAV2 infected cells was over 30%. CONCLUSION： The viral vector of rAAV2-EGFP-U6-shRNA （ VEGF165 ） is successfully prepared. This viral vector will serve to interpret the role of VEGF in biological events after cerebral ischemic injury by specially inhibiting the expression of VEGF gene.