IRF5基因慢病毒表达载体的构建及其在巨噬细胞RAW264.7中的表达

doi:10.13481/j.1671-587x.20160307

摘要/Abstract

摘要：

目的: 构建干扰素凋节因子5(IRF5)慢病毒表达载体LV11-cmv-neo-IRF5并转染巨噬细胞RAW264.7,观察IRF5巨噬细胞RAW264.7中的表达。方法: 采用基因重组技术,将PCR技术钓取的目的基因IRF5片段克隆至线性化慢病毒表达载体LV11中,重组载体经PCR、双酶切和测序鉴定构建成功后,采用脂质体转染技术将表达质粒和包装质粒共转染293FT细胞,获得携带IRF5基因的重组慢病毒;收集病毒上清液感染小鼠巨噬细胞RAW264.7,用G418将非浸染的细胞致死,获得纯净的浸染细胞,即RAW264.7-IRF5细胞(实验组),并将RAW264.7-NC细胞作为对照(对照组)。分别采用RT-qPCR和Western blotting法检测2组细胞中IRF5mRNA和蛋白的表达水平。结果: 重组慢病毒质粒LV11-cmv-neo-IRF5经PCR和酶切法鉴定构建成功;病毒上清液感染RAW264.7细胞并经G418筛选获得阳性细胞;RT-qPCR法检测,与对照组比较,实验组细胞中IRF5mRNA表达水平升高(P<0.001);Western blotting法检测,实验组细胞中IRF5蛋白的表达水平明显高于对照组。结论: 成功构建携带IRF5基因的慢病毒表达载体LV11-cmv-neo-IRF5,IRF5基因在巨噬细胞RAW264.7中稳定表达。

关键词: 干扰素调节因子5, 慢病毒表达载体, 动脉粥样硬化, 巨噬细胞

Abstract:

Objective: To construct interferon regulatory factor 5(IRF5) gene lentiviral vector LV11-cmv-neo-IRF5 and transfect it into macrophages RAW264.7, and to observe its expression in RAW264.7 cells. Methods: The target gene IRF5 gragment was fished by PCR and cloned into linearized lentiviral vector LV11 by using DNA recombinant technique. The recombinant vector was identified by PCR, double enzyme digestion,and sequencing and the 293T cells were transfected with the lipofectin reagent for lentiviral particles packaged with expression and packing plasmids. The supernatant of lentiviral was collected and infected into the macrophages RAW264.7. After screening the cells, which were not infected with G418, the positive infected cells(RAW264.7-IRF5)were obtained (experiment group). Meanwhile, the RAW264.7-NC cells was regarded as controls(control group).The expression levels of IRF5 mRNA and protein in RAW264.7 cells in two groups were examined by RT-qPCR and Western blotting methods. Results: The PCR and enzyme digestion analysis results confirmed that the lentiviral vector LV11-cmv-neo-IRF5 was successfully constructed. The RAW264.7 cells were infected with virus supernatant and the positive cells were obtained by G418 screening.The RT-qPCR results showed that compared with control group, the expression level of IRF5 mRNA in the RAW264.7 cells in experiment group was increased (P<0.01);the Western blotting results showed that compared with control group,the IRF5 protein epression level in the RAW264.7 cells in experiment group was increased. Conclusion: The lentiviral vector LV11-cmv-neo-IRF5 containing IRF5 gene is successfully constructed,and the IRF5 gene can express stably in the macrophages RAW264.7.

Key words: interferon regulatory factor 5, lentiviral vector, atherosclerosis, macrophages

中图分类号:

麦莉, 韦建瑞, 华兴. IRF5基因慢病毒表达载体的构建及其在巨噬细胞RAW264.7中的表达[J]. 吉林大学学报(医学版), 2016, 42(03): 452-456.

MAI Li, WEI Jianrui, HUA Xing. Construction of IRF5 gene lentiviral vector and its expression in macrophages RAW264.7[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 452-456.

参考文献

[1] 邢宝鹏,纪莉. 冠状动脉粥样硬化性心脏病相关危险因素及临床特点分析[D].长春:吉林大学,2012.

[2] 黄燕. 炎性因子在动脉粥样硬化形成中的作用[D]. 天津:天津医科大学, 2012.

[3] Shirai T, Hilhorst M, Harrison DG,et al. Macrophages in vascular inflammation-From atherosclerosis to vasculitis[J]. Autoimmunity, 2015,48(3): 139-151.

[4] Eames HL, Saliba DG, Krausgruber T,et al. KAP1/TRIM28: an inhibitor of IRF5 function in inflammatory macrophages[J]. Immunobiology, 2012,217(12):1315-1324.

[5] Kim K, Cho SK, Han TU,et al. A redundant epistatic interaction between IRF5 and STAT4 of the type Ⅰ interferon pathway in susceptibility to lupus and rheumatoid arthritis[J]. Lupus,2013,22(13):1336-1340.

[6] Maalej A, Hamad MB, Rebai A, et al. Association of IRF5 gene polymorphisms with rheumatoid arthritis in a Tunisian population[J]. Scand J Rheumatol, 2008,37(6):414-418.

[7] Gestermann N, Koutero M, Belkhir R, et al. Methylation profile of the promoter region of IRF5 in primary Sjogren's syndrome[J]. Eur Cytokine Netw, 2012,23(4):166-172.

[8] Krausgruber T, Saliba D, Ryzhakov G, et al. IRF5 is required for late-phase TNF secretion by human dendritic cells[J]. Blood,2010,115(22):4421-4430.

[9] Krausgruber T, Blazek K, Smallie T, et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses[J]. Nat Immunol, 2011,12(3):231-238.

[10] Mantovani A, Sozzani S, Locati M, et al.Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes[J]. Trends Immunol, 2002,23(11),549-555.

[11] Chinetti-Gbaguidi G, Baron M, Bouhlel MA, et al. Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways[J]. Circ Res, 2011, 108(8) :985-995.

[12] Malarstig A, Sigurdsson S, Eriksson P, et al. Variants of the interferon regulatory factor 5 gene regulate expression of IRF5 mRNA in atherosclerotic tissue but are not associated with myocardial infarction[J]. Arterioscler Thromb Vasc Biol, 2008,28(5):975-982.

[13] Suzuki M, Pritchard DK, Becker L, et al. High-density lipoprotein suppresses the type I interferon response,a family of potentantiviral immunoregulators,in macrophages challenged with lipopolysaccharide[J].Circulation,2010,122(19):1919-1927.

[14] Takaoka A,Yanai H,Kondo S,et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors[J]. Nature,2005,434(7030): 243-249.

[15] Courties G, Heidt T, Sebas M, et al. In vivo silencing of the transcription factor IRF5 reprograms the macrophage phenotype and improves infarct healing[J]. J Am Coll Cardiol, 2014,63(15): 1556-1566.

[16] Xu WD, Pan HF, Xu Y,et al. Interferon regulatory factor 5 and autoimmune lumps[J]. Expert Rev Mol Med, 2013,15:e6.