小鼠FoxP3基因腺相关病毒的制备与鉴定

目的:构建携带小鼠FoxP3基因的重组腺相关病毒,并检测其在NIH3T3细胞的表达情况。方法:将FoxP3通过内部核糖体进入位点(IRES)与报告基因增强型绿色荧光蛋白连接,构建同时含有目的基因和报告基因的重组腺相关病毒(rAAV)表达质粒,通过磷酸钙沉淀法共转染HEK293细胞,收获并通过肝素亲和层析法纯化病毒,并对病毒纯度和滴度进行鉴定。利用携带FoxP3基因的重组病毒体外感染小鼠NIH3T3细胞,体外观察感染效率和目的基因转录情况。结果:包装成功的rAAV/FoxP3经纯化后获得了高纯度的rAAV/FoxP3,实时定量PCR检测rAAV/FoxP3的病毒滴度达到5.0×1012vg/mL,体外感染NIH3T3细胞,流式细胞术测定感染效率可达92.88%,实时PCR测定细胞内存在高水平FoxP3mRNA。结论:获得携带FoxP3-IRES-EGFP的rAAV,并可有效感染NIH3T3细胞,为进一步研究FoxP3的生物学功能提供了有效工具。

Construction and identification of the recombination adeno-associated virus carrying murine FoxP3 gene

AIM: To construct the recombination adeno-associated virus (rAAV) carrying murine forkhead box P3 (FoxP3) gene, and then detect the expression in NIH3T3 cells. METHODS: The recombination adeno-associated virus (rAAV) vector containing internal ribosome entry site which could coordinate the expression of FoxP3 gene and enhance green fluorescent protein gene was constructed. HEK293 cells were transfected by the transfection cocktail containing the constructed rAAV vector, trans plasmid and helper plasmid by the calcium phosphate method. The infectious rAAV carrying FoxP3 gene (rAAV/FoxP3) suspensions were harvested and purified by heparin affinity chromatography. The purity and titre of rAAV were detected by SDS-PAGE electrophoresis and real-time PCR, respectively. NIH3T3 cells were transfected by rAAV, the transfection efficiency was analyzed by flow cytometry and FoxP3 mRNA expression was detected by real-time PCR. RESULTS: The titre of the infectious rAAV was 5x 10(12) vg/mL by real-time PCR analysis. After NIH3T3 cells were transfected, the transfection efficiency of the infectious rAAV/FoxP3 was up to 92.88% and high level of FoxP3 mRNA was detected. CONCLUSION: rAAV carrying FoxP3 gene was successfully constructed and expressed in NIH3T3 cells, which will be of benefit to further researches on the function of FoxP3.