Dectin-1慢病毒过表达载体的构建与鉴定

Constructionand Identification of Lentivirus Vector of Dectin-1 Gene

目的通过构建Dectin-1慢病毒过表达载体,感染巨噬细胞RAW264.7,建立Dectin-1过表达的巨噬细胞模型。方法根据小鼠Dectin-1基因序列设计引物,经PCR扩增目的基因后克隆至载体pLenti6.3-IRES2-EGFP/V5 DEST(MCS),获得重组质粒pLenti6.3-dectin-1-IRES-EGF。经和包装质粒Mix共同转染293T细胞,收获慢病毒,利用293T细胞大量扩增慢病毒并进行慢病毒的滴度测定,根据MOI值感染巨噬细胞RAW264.7,经荧光显微镜和荧光定量PCR鉴定感染慢病毒组和感染空病毒组中Dectin-1基因的表达。结果 PCR和测序结果证明已成功构建pLenti6.3-dectin-1-IRES-EGFP,成功包装慢病毒,计算慢病毒滴度为3.12×10~9 Tu/mL,成功感染巨噬细胞RAW264.7,荧光显微镜下可见绿色荧光,RT-PCR检测到感染重组慢病毒组Dectin-1表达量是感染空病毒组的1 275倍(P<0.01)。结论成功构建Dectin-1基因过表达的巨噬细胞模型,为进一步研究Dectin-1基因在巨噬细胞对抗真菌感染中的作用打下基础。

Objective To establish a Dectin-1 gene overexpressed macrophage model by constructing a lentiviral vector of Dectin-1,and infecting macrophage RAW264.7.Methods Primers were designed according to the sequence of mouse Dectin-1 gene.The PCR-amplified products were cloned into vector pLenti6.3-IRES2-EGFP/V5 DEST(MCS),and the recombinant lentiviral plasmid pLenti6.3-dectin-1-IRES-EGF were made.The recombinant lentiviral plasmid and packaging plasmid mix were transfected into 293 T cells.Lentivirus was amplified in the 293 T cells,followed by titration.The macrophage RAW264.7 was transfected according to MOI value.Fluorescence microscopy and Real-time PCR were used to determine the expression of Dectin-1 gene.Results Both PCR and sequencing confirmed that the recombinant plasmid pLenti6.3-dectin-1-IRES-EGF was properly constructed.The lentivirus was successfully packaged with titer of 3.12×10~9 Tu/mL.The macrophage RAW264.7 was effectively transfected,as indicated by presence of green fluorescence in the cells under microscope.The real-time PCR assay showed that the expression levels of Dectin-1 gene in macrophages transfected with recombinant lentivirus were 1 275 folds of that transfected with empty lentivirus(P<0.01).Conclusion A Dectin-1 gene overexpressed macrophage model was successfully established,providing a foundation for further research in the role of dectin-1 gene in macrophages against fungi infections.