【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(P<0.05)。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。更多还原

【Abstract】 Objective To construct a eukaryotic expression plasmid for recombinant mouse Toll-like receptor 7(rm TLR7)gene and analyze its effect on the immune function of mouse bone marrow derived dendritic cells(Bm-DCs).Methods Primary DCs were prepared with the splenocytes of C57 BL / 6 mice,from which total RNA was extracted and reversely transcribed to c DNA.TLR7 gene was amplified using the c DNA as a template and cloned into vector pc DNA3.1(+),and the constructed recombinant plasmid pc DNA3.1-TLR7 was transfected to mouse DCs in mediation of Lipofectamine 2000.Positive clones were screened with G418 and determined for m RNA transcription and protein expression level of TLR7 by real-time PCR and Western blot respectively.Meanwhile,the expressions of co-stimulators CD80,CD86 and MHCⅡ in the transfected DCs were determined by flow cytometry,while the secretion levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)by ELISA kit.Results PCR and restriction analysis demonstrated that recombinant plasmid pc DNA3.1-TLR7 was constructed correctly.Both m RNA transcription and protein expression of TLR7 were observed in DCs 12,24,48 and 72 h,of which the levels reached the peak values 48 h,after transfection.The expression levels of CD80,CD86 and MHC Ⅱ and secretion levels of IL-6 and TNF-α in DCs increased significantly 48 h after transfection(P < 0.05).Conclusion The eukaryotic expression plasmid for recombinant TLR7 was successfully constructed,which enhanced the immune function of DCs significantly.更多还原