GFP重组腺相关病毒报告病毒的构建及其在NIH3T3细胞中的表达

目的构建以绿色荧光蛋白(GFP)为报告基因的重组腺相关病毒载体和报告病毒,并观察其在真核细胞的表达。方法采用DNA重组技术将GFP基因片段亚克隆到pss HGCMV载体的EcoRⅠ和BamHⅠ位点,构建表达GFP重组腺相关病毒载体pss HGCMV-GFP。以此载体转染143细胞进行GFP重组腺相关病毒包装,产生重组报告病毒颗粒并作纯化。用地高辛标记的CMV polyA片段作为探针,采用斑点杂交方法检测重组病毒的物理滴度。采用氯化钙法体外转染培养的NI H3T3细胞,在活细胞状态下用荧光显微镜直接观察其在细胞中的表达。结果成功构建了GFP重组腺相关病毒载体和报告病毒—VssCMV-GFP,所获病毒的滴度为3.16×1012病毒颗粒/mL。重组腺相关病毒VssCMV-GFP感染NI H3T3细胞后48h开始出现绿色荧光,并逐渐增强,至120h荧光更加集中且很强。结论VssCMV-GFP对真核细胞具有感染能力,并且GFP可以在活细胞内有效表达,提示该重组腺相关病毒可应用于介导基因转移真核细胞进行基因治疗。

Construction of GFP reconbinant AAV- reporter virus and its expression in NIH 3T3 cell

Objective To construct a recombinant AAV reporter virus expressing GFP and observe its expression in NIH 3T3 cell. Methods The resulting gene of GFP was subcloned into the site EcoRⅠand BamHⅠof vector pssHGCMV, and a recombinant adeno-associated virus (rAAV) vector pssHGCMV-GFP expressing GFP was constructed by using DNA recombinant technique. The 143 cells were transfected with the vector and the rAAV stock expressing GFP was packaged, produced and purified. The titre of the rAAV stock was measured by quantitative dot blot hybridization. NIH 3T3 cells were infected with the rAAV and the fluorescence was detected in alive cells to confirm successful infection and the expression of exogenous gene in NIH 3T3 cell line. Results A GFP-expressing rAAV vector VssCMV-GFP was successfully constructed. The titer of the VssCMV-GFP was 3.16×10~(12) particles/mL. GFP was initially expressed in the NIH 3T3 cells 48h following infected by VssCMV-GFP and subsequently it gradually increased. The green fluorescence in the cells was the most apparent 120h after infected. Conclusion VssCMV-GFP can infect the eukaryotic cells and GFP can be expressed in the alive cells.It is suggested that rAAV-mediated transfer of the gene to the eukaryotic cells can serve as a delivery system of gene therapy.