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

作　　者：沈林霞 徐红艳 刘栋华[1] SHEN Linxia;XU Hongyan;LIU Donghua(Department of Dermatology and Venereology,First Affiliated Hospital of Guangxi Medical University,Nannning 530021 ,China)

机构地区：[1]广西医科大学第一附属医院皮肤性病科广西艾滋病防治研究重点实验室,广西南宁530021

出　　处：《中国皮肤性病学杂志》2019年第5期507-512,共6页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金(81760564)

摘　　要：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.

关 键 词：慢病毒 DECTIN-1 真菌 

分 类 号：R756.6[医药卫生—皮肤病学与性病学] Q782[医药卫生—临床医学]