单纯疱疹病毒Ⅱ型CTCF结合位点抑制启动子功能

CTCF-binding Sites in Herpes Simplex Virus Type Ⅱ Display Promoter-blocking Activity

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。

To find the locations of CTCF-binding motifs in herpes simplex virus type 2 （HSV-2） and identi- fy their functions in regulation of genome expression. HSV-1 and HSV-2 were compared. Bioinformatics a- nalysis of HSV-2 genomes and Jaspar analysis coupled with sequence evaluation were used to find the CTCF-binding motifs. The predicted binding motifs were amplified by polymerase chain reaction and em- bedded into pGL3-promoter plasmids. The recombinant plasmids were transfected into human embryonic kidney cells （293T）. Then, dual luciferase reporter assays were utilized to identify the functions of these motifs. We predicted that CTCF-binding motifs would be located in the latent-associated transcript （LAT） upstream （CTUL）, downstream （CTa~ m） and intron （CTRL）. Double digestion and sequencing con- firmed these recombinant plasmids had been constructed successfully. Lueiferase reporter assays revealed that pGL3-promoter-CTRL and pGL3-promoter-CTa＇m, containing the LAT intron and downstream-pre- dicted motifs, respectively, expressed lower lueiferase activities than the pGL3-promoter （P〈0. 001）, but did not silenced luciferase gene expression compared with pGL3-bisaee. The pGL3-promoter-CTUL group showed a significant difference compared with the pGL3-promoter group （P〉0. 05）. CTCF-binding motifs are located in the LAT downstream and intron, and display promoter-blocking activity that may play an important part in HSV-2 latency.