单纯疱疹病毒Ⅱ型CTCF结合位点抑制启动子功能

发布时间：2018-03-26 11:07

本文选题：单纯疱疹病毒Ⅱ型(HSV-)　切入点：潜伏复发机制　出处：《病毒学报》2017年04期

【摘要】：为了找到CTCF在单纯疱疹病毒Ⅱ型(HSV-2)中的结合位点,并探讨其基因表达调控作用。对比HSV-1与HSV-2全基因组中CTCF结合序列区别;生物信息学分析HSV-2全基因组;Jaspar在线预测CTCF结合位点;依据CTCF结合的序列特点预测结合位置;PCR扩增预测位点并插入pGL3-promoter构建重组载体;重组载体转染人胚胎肾细胞(293T)双荧光检测验证预测位点转录调控效应。预测得到三个CTCF结合位点分别位于潜伏相关转录本(LAT)上游(CTUL)、下游(CTa’m)及内含子(CTRL)位置;扩增目的片段并构建重组载体,双酶切及测序验证成功;双荧光检测显示LAT内含子(pGL3-promoter-CTRL)及下游(pGL3-promoter-CTa’m)结合位点重组载体转染组与pGL3-promoter载体转染组相比,荧光强度明显减弱,差异有统计学意(P0.001),但与pGL3-basic组相比,并没有完全沉默荧光霉素的表达;上游结合位点重组载体(pGL3-promoter-CTUL)转染组与pGL3-promoter相比无明显差异(P0.05)。HSV-2LAT序列内含子及下游序列上存在CTCF结合位点,具有减弱基因启动子功能的效应,可能在HSV-2维持潜伏中发挥作用。
[Abstract]:In order to find the binding site of CTCF in HSV-2 of herpes simplex virus (HSV-2) and to investigate its gene expression regulation, the difference of CTCF binding sequence between HSV-1 and HSV-2 genome was compared, and the bioinformatics analysis of HSV-2 genome-wide Jaspar was used to predict CTCF binding site online. According to the sequence characteristics of CTCF binding, the predicted site was amplified by PCR and inserted into pGL3-promoter to construct recombinant vector. The transcriptional effects of the predicted sites were verified by double fluorescence detection of the recombinant vector transfected into human embryonic kidney cells (293T). The three CTCF binding sites were predicted to be located at the CTCF sites in the upstream, downstream, and intron CTRL positions of the latent related transcripts (LATs), respectively. The target fragment was amplified, the recombinant vector was digested and sequenced successfully, the fluorescence intensity of the recombinant vector transfected with LAT intron pGL3-promoter-CTRL and downstream pGL3-promoter-CTam) was significantly lower than that of the pGL3-promoter vector transfection group, and the fluorescence intensity of the recombinant vector transfected group was significantly lower than that of the pGL3-promoter vector transfection group, and the fluorescence intensity of the recombinant vector was significantly lower than that of the pGL3-promoter vector transfection group. Compared with pGL3-basic group, the expression of fluorescein was not completely silenced, and there was no significant difference between the upstream binding site recombinant vector pGL3-promoter-CTULL group and pGL3-promoter. There were CTCF binding sites in intron and downstream sequence of HSV-2LAT sequence. It has the effect of attenuating the function of gene promoter and may play a role in the maintenance of HSV-2 latency.
【作者单位】：华南理工大学生物科学与工程学院;广州军区广州总医院皮肤科;
【基金】：国家自然科学基金(项目号:81371749),题目:CTCF在单纯疱疹病毒Ⅱ型潜伏复发中的作用研究~~
【分类号】：R373.11

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