本文選題：單純皰疹病毒Ⅱ型(HSV-)　切入點：潛伏復發(fā)機制　出處：《病毒學報》2017年04期

【摘要】：為了找到CTCF在單純皰疹病毒Ⅱ型(HSV-2)中的結合位點,并探討其基因表達調控作用。對比HSV-1與HSV-2全基因組中CTCF結合序列區(qū)別;生物信息學分析HSV-2全基因組;Jaspar在線預測CTCF結合位點;依據CTCF結合的序列特點預測結合位置;PCR擴增預測位點并插入pGL3-promoter構建重組載體;重組載體轉染人胚胎腎細胞(293T)雙熒光檢測驗證預測位點轉錄調控效應。預測得到三個CTCF結合位點分別位于潛伏相關轉錄本(LAT)上游(CTUL)、下游(CTa’m)及內含子(CTRL)位置;擴增目的片段并構建重組載體,雙酶切及測序驗證成功;雙熒光檢測顯示LAT內含子(pGL3-promoter-CTRL)及下游(pGL3-promoter-CTa’m)結合位點重組載體轉染組與pGL3-promoter載體轉染組相比,熒光強度明顯減弱,差異有統(tǒng)計學意(P0.001),但與pGL3-basic組相比,并沒有完全沉默熒光霉素的表達;上游結合位點重組載體(pGL3-promoter-CTUL)轉染組與pGL3-promoter相比無明顯差異(P0.05)。HSV-2LAT序列內含子及下游序列上存在CTCF結合位點,具有減弱基因啟動子功能的效應,可能在HSV-2維持潛伏中發(fā)揮作用。
[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.
【作者單位】： 華南理工大學生物科學與工程學院;廣州軍區(qū)廣州總醫(yī)院皮膚科;
【基金】：國家自然科學基金(項目號:81371749),題目:CTCF在單純皰疹病毒Ⅱ型潛伏復發(fā)中的作用研究~~
【分類號】：R373.11

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