重组Toll样受体7真核表达质粒的构建及其对髓样树突状细胞免疫功能的影响

发布时间：2018-01-08 03:29

本文关键词：重组Toll样受体7真核表达质粒的构建及其对髓样树突状细胞免疫功能的影响　出处：《中国生物制品学杂志》2016年05期 　论文类型：期刊论文

【摘要】：目的构建重组Toll样受体7(Toll-ike receptor 7,TLR7)真核表达质粒,并分析其对髓样树突状细胞(dendritic cell,DC)免疫功能的影响。方法用C57BL/6小鼠脾细胞制备原代DC,提取DC总RNA,以其逆转录合成的c DNA为模板扩增TLR7基因,克隆至载体pc DNA3.1(+)中,构建真核表达质粒pc DNA3.1-TLR7。经脂质体Lipofectamine2000将真核表达质粒转染至小鼠DC,经G418筛选阳性克隆。分别采用Real-time PCR及Western blot法检测转染细胞中TLR7基因m RNA转录及蛋白的表达水平;同时采用流式细胞术及细胞因子试剂盒检测转染细胞表面共刺激分子CD80、CD86和MHCⅡ的表达及分泌白细胞介素-6(interleukins-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平。结果经PCR及双酶切鉴定证明重组真核表达质粒pc DNA3.1-TLR7构建正确。转染12、24、48及72 h的DC中均可见TLR7基因的转录及蛋白的表达,且48 h时的转录及表达水平最高。转染48 h后,DC表面共刺激因子CD80、CD86和MHCⅡ的表达水平及其分泌IL-6和TNF-α的水平均显著提高(P0.05)。结论成功建立了重组TLR7真核表达质粒,且其可明显提高DC的免疫功能。
[Abstract]:Objective to construct a recombinant Toll like receptor 7 (Toll-ike receptor 7, TLR7) eukaryotic expression plasmid, and analysis of myeloid dendritic cells (dendritic, cell, DC) immune function. Methods C57BL/6 mice spleen cells were prepared from the primary DC, DC total RNA extraction, reverse transcriptase synthesis with its C DNA as template TLR7 gene was amplified and cloned into the vector PC DNA3.1 (+) and the eukaryotic expression plasmid PC was constructed by liposome DNA3.1-TLR7. Lipofectamine2000 eukaryotic expression plasmid was transfected into DC mice. The positive clones were selected by G418. The expression level of Real-time PCR and Western blot were used to assay TLR7 cells transfected with M gene transcription and protein RNA at the same time; using flow cytometry and cell ELISA Kit transfected cell surface costimulatory molecules CD80, CD86 and MHC II expression and secretion of interleukin -6 (interleukins-6, IL-6), tumor necrosis factor alpha (tumor necrosis fac Tor- alpha, alpha TNF-) level. The results by PCR and double enzyme digestion indicated that recombinant eukaryotic expression plasmid PC DNA3.1-TLR7 was constructed correctly. The expression of mRNA and protein were observed in TLR7 gene transfection of 12,24,48 and 72 h in DC, and 48 h transcription and expression of the highest level. After 48 h, DC the surface CO stimulatory factor CD80 expression level of CD86 and MHC II and the level of secretion of IL-6 and TNF- alpha were significantly increased (P0.05). Conclusion the recombinant eukaryotic expression plasmid of TLR7, and it can significantly improve the immune function of DC.

【作者单位】：南方医科大学南方医院实验动物研究中心;南方医科大学南方医院肾内科;
【基金】：广东省科技计划项目(2014A030304024);广东省科技计划项目(2013B060300012) 南方医院院长基金项目(201213006)
【分类号】：R392.12
【正文快照】： 树突状细胞(dendritic cell,DC)是体内功能最强大的抗原提呈细胞,主要依赖其表面的Toll样受体(Toll-like receptor,TLR)识别病原微生物,是连接固有免疫应答和特异性免疫的桥梁[1]。TLR7是一种表达于细胞内涵体膜表面的跨膜信号转导受体,可识别某些小分子的抗病毒化合物和病毒

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