[1]沈林霞,徐红艳,刘栋华.Dectin-1慢病毒过表达载体的构建与鉴定[J].中国皮肤性病学杂志,2019,(05):507-512.[doi:10.13735/j.cjdv.1001-7089.201811065]
 SHEN Linxia,XU Hongyan,LIU Donghua.Constructionand Identification of Lentivirus Vector of Dectin-1 Gene[J].The Chinese Journal of Dermatovenereology,2019,(05):507-512.[doi:10.13735/j.cjdv.1001-7089.201811065]
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Dectin-1慢病毒过表达载体的构建与鉴定
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《中国皮肤性病学杂志》[ISSN:1001-7089/CN:61-1197/R]

卷:
期数:
2019年05期
页码:
507-512
栏目:
论著
出版日期:
2019-04-01

文章信息/Info

Title:
Constructionand Identification of Lentivirus Vector of Dectin-1 Gene
文章编号:
1001-7089(2019)05-0507-06
作者:
沈林霞徐红艳刘栋华
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
Author(s):
SHEN Linxia XU Hongyan LIU Donghua
(Department of Dermatology and Venereology,First Affiliated Hospital of Guangxi Medical University, Nannning 530021,China)
关键词:
慢病毒Dectin-1真菌
Keywords:
LentivirusDectin-1Fungi
分类号:
R 756.6;Q 782
DOI:
10.13735/j.cjdv.1001-7089.201811065
文献标志码:
A
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3.12×109 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。
Abstract:
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 293T cells.Lentivirus was amplified in the 293T 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.ResultsBoth 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 3.12×109 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).ConclusionA 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.

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备注/Memo

备注/Memo:
[基金项目]国家自然科学基金(81760564)
[作者单位]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
[通讯作者]刘栋华,E-mail: ldhgxmu@163.com
更新日期/Last Update: 2019-04-15