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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(P<0.001),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(P>0.05)。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。
In order to find out the binding site of CTCF in herpes simplex virus type 2 (HSV-2), and to explore its gene expression regulation. The whole genome of HSV-1 was compared with that of HSV-1 in HSV-2 genome. HSV-2 whole genome was bioinformatically analyzed. Jaspar was used to predict CTCF binding sites on the line. The binding sites were predicted based on the sequence characteristics of CTCF binding. And inserted into pGL3-promoter to construct the recombinant vector. The recombinant vector was transfected into human embryonic kidney cells (293T) double-fluorescent detection to verify the site-specific transcriptional regulation. Three CTCF binding sites were predicted to be located upstream (CTUL), downstream (CTa’m) and intron (CTRL) of the LAT, respectively. The target fragment was amplified and the recombinant vector was constructed. The results of sequencing showed that fluorescence was detected by double-fluorescence assay. Compared with pGL3-promoter vector transfected group, the expression of pGL3-promoter-CTRL and pGL3-promoter-CTa’m (P <0.001). However, compared with pGL3-basic group, the expression of phosphomycin was not completely silenced. The expression of pGL3-promoter-CTUL transfection group Compared with pGL3-promoter, there was no significant difference (P> 0.05). The presence of a CTCF binding site on the HSV-2 LAT sequence introns and downstream sequences has the effect of attenuating gene promoter function and may play a role in the maintenance latency of HSV-2.