小鼠盐皮质激素受体基因shRNA慢病毒干扰系统的建立与鉴定
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摘要
【目的】应用RNA干扰技术,构建小鼠盐皮质激素受体(mineralcorticoid receptor,MR)基因重组慢病毒载体并鉴定。【方法】根据本室已经筛选的有效RNA干扰序列和阴性对照序列,合成单链引物并经退火形成双链寡核苷酸序列,连接经EcoR I和BamH I双酶切线性化的pLVX-shRNA-Puro慢病毒质粒载体中,提取质粒DNA并经测序验证。测序正确后,在Lipofectamine~3000的介导下,与慢病毒包装辅助质粒psPAX2、pMD2.G共转染293T细胞,收集病毒上清经浓缩后感染小鼠巨噬细胞RAW264.7,流式细胞术评价其滴度及感染复数,实时定量PCR法鉴定MR shRNA慢病毒的干扰效率。【结果】成功构建了重组质粒pLVX-shRNA-MR,经DNA测序验证载体构建成功。流式细胞术测定重组慢病毒的感染复数大约为100,实时定量PCR法和Western Blot法检测干扰效率大约为75%左右。【结论】成功构建出小鼠MR基因shRNA慢病毒干扰系统。
【Objective】To construct and identify the lentiviral vector of shRNA interference targeting mouse mineralcorticoid receptor gene.【Methods】RNAi target sequences were designed and synthesized focused on mouse mineralcorticoid receptor gene sequence,and cloned into the lentiviral vector pLVX-shRNA-Puro by restriction endonuclease EcoR I and BamH I double digestion and T4 DNA ligase ligation.After transformation into competent E.coli,the clones were identified by DNA sequencing.The recombinant plasmid and the two packaging plasmids were co-transfected into the human embryonic kidney 293 T cells by Lipofectamine3000~ to produce lentiviral particles.Then recombinant lentivirus was harvested to infect RAW264.7 cells.The titer and multiplicity of infection were evaluated by flow cytometry and the expression of MR mRNA in RAW264.7 cells was measured by Real-time PCR.【Results】The lentiviral RNAi vector pLVX-shRNA-Puro-MR was successfully constructed and verified by DNA sequencing.The multiplicity of infection of lentiviral vector of shRNA interference is 100 by FCM.The recombinant lentivirus significantly inhibited the expression of MR by 75% compared with negative control(NC) through Real-time PCR and Western blotting.【Conclusion】The recombinant lentiviral vector of shRNA targeting MR has been successfully constructed.
【Objective】To construct and identify the lentiviral vector of shRNA interference targeting mouse mineralcorticoid receptor gene.【Methods】RNAi target sequences were designed and synthesized focused on mouse mineralcorticoid receptor gene sequence,and cloned into the lentiviral vector pLVX-shRNA-Puro by restriction endonuclease EcoR I and BamH I double digestion and T4 DNA ligase ligation.After transformation into competent E.coli,the clones were identified by DNA sequencing.The recombinant plasmid and the two packaging plasmids were co-transfected into the human embryonic kidney 293 T cells by Lipofectamine3000~ to produce lentiviral particles.Then recombinant lentivirus was harvested to infect RAW264.7 cells.The titer and multiplicity of infection were evaluated by flow cytometry and the expression of MR mRNA in RAW264.7 cells was measured by Real-time PCR.【Results】The lentiviral RNAi vector pLVX-shRNA-Puro-MR was successfully constructed and verified by DNA sequencing.The multiplicity of infection of lentiviral vector of shRNA interference is 100 by FCM.The recombinant lentivirus significantly inhibited the expression of MR by 75% compared with negative control(NC) through Real-time PCR and Western blotting.【Conclusion】The recombinant lentiviral vector of shRNA targeting MR has been successfully constructed.
引文
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[10]姬文婕,胡道川,陈雪芬,等.干扰盐皮质激素受体基因对RAW264.7细胞增殖和凋亡的影响[J].中国病理生理杂志,2012,28(12):2261-2265.
[11]Ji WJ,Ma YQ,Zhou X,et al.Spironolactone attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in circulating and alveolar compartments[J].PLo S ONE,2013,8(11):e81090.
[12]Ji WJ,Ma YQ,Zhang X,et al.Inflammatory monocyte/macrophage modulation by liposome-entrapped spironolactone ameliorates acute lung injury in mice[J].Nanomedicine(Lond),2016,11(11):1393-1406.
[2]Crochemore C,Lu J,Wu Y,et al.Direct targeting of hippocampal neurons for apoptosis by glucocorticoids is reversible by mineralocorticoid receptor activation[J].Mol Psychiatry,2005,10(8):790-798.
[3]Ishizawa K,Izawa Y,Ito H,et al.Aldosterone stimulates vascular smooth muscle cell proliferation via big mitogen-activated protein kinase 1 activation[J].Hypertension,2005,46(4):1046-1052.
[4]Ouvrard-Pascaud A,Sainte-Marie Y,Bénitah JP,et al.Conditional mineralocorticoid receptor expression in the heart leads to life-threatening arrhythmias[J].Circulation,2005,111(23):3025-3033.
[5]袁婺洲,吴秀山.RNAi机制研究的最新进展[J].生命科学研究,2003,7(1):8-14.
[6]Lois C,Hong EJ,Pease S,et al.Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J].Science,2002,295(5556):868-872.
[7]Pfeifer A,Ikawa M,Dayn Y,et al.Transgenesis by lentiviral vectors:lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos[J].Proc Natl Acad Sci USA,2002,99(4):2140-2145.
[8]Mc Curley A,Jaffe IZ.Mineralocorticoid receptors in vascular function and disease[J].Mol Cell Endocrinol,2012,350(2):256-265.
[9]许明,杨汉东,闵新文,等.安体舒通改善自发性高血压大鼠心肌纤维化[J].中国病理生理杂志,2008,24(7):1281-1285.
[10]姬文婕,胡道川,陈雪芬,等.干扰盐皮质激素受体基因对RAW264.7细胞增殖和凋亡的影响[J].中国病理生理杂志,2012,28(12):2261-2265.
[11]Ji WJ,Ma YQ,Zhou X,et al.Spironolactone attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in circulating and alveolar compartments[J].PLo S ONE,2013,8(11):e81090.
[12]Ji WJ,Ma YQ,Zhang X,et al.Inflammatory monocyte/macrophage modulation by liposome-entrapped spironolactone ameliorates acute lung injury in mice[J].Nanomedicine(Lond),2016,11(11):1393-1406.