本文選題：單純皰疹病毒Ⅱ型(HSV-)　切入點(diǎn)：潛伏復(fù)發(fā)機(jī)制　出處：《病毒學(xué)報(bào)》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(jié)合位點(diǎn),并探討其基因表達(dá)調(diào)控作用。對(duì)比HSV-1與HSV-2全基因組中CTCF結(jié)合序列區(qū)別;生物信息學(xué)分析HSV-2全基因組;Jaspar在線預(yù)測(cè)CTCF結(jié)合位點(diǎn);依據(jù)CTCF結(jié)合的序列特點(diǎn)預(yù)測(cè)結(jié)合位置;PCR擴(kuò)增預(yù)測(cè)位點(diǎn)并插入pGL3-promoter構(gòu)建重組載體;重組載體轉(zhuǎn)染人胚胎腎細(xì)胞(293T)雙熒光檢測(cè)驗(yàn)證預(yù)測(cè)位點(diǎn)轉(zhuǎn)錄調(diào)控效應(yīng)。預(yù)測(cè)得到三個(gè)CTCF結(jié)合位點(diǎn)分別位于潛伏相關(guān)轉(zhuǎn)錄本(LAT)上游(CTUL)、下游(CTa’m)及內(nèi)含子(CTRL)位置;擴(kuò)增目的片段并構(gòu)建重組載體,雙酶切及測(cè)序驗(yàn)證成功;雙熒光檢測(cè)顯示LAT內(nèi)含子(pGL3-promoter-CTRL)及下游(pGL3-promoter-CTa’m)結(jié)合位點(diǎn)重組載體轉(zhuǎn)染組與pGL3-promoter載體轉(zhuǎn)染組相比,熒光強(qiáng)度明顯減弱,差異有統(tǒng)計(jì)學(xué)意(P0.001),但與pGL3-basic組相比,并沒有完全沉默熒光霉素的表達(dá);上游結(jié)合位點(diǎn)重組載體(pGL3-promoter-CTUL)轉(zhuǎn)染組與pGL3-promoter相比無明顯差異(P0.05)。HSV-2LAT序列內(nèi)含子及下游序列上存在CTCF結(jié)合位點(diǎn),具有減弱基因啟動(dòng)子功能的效應(yīng),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(xué)生物科學(xué)與工程學(xué)院;廣州軍區(qū)廣州總醫(yī)院皮膚科;
【基金】：國家自然科學(xué)基金(項(xiàng)目號(hào):81371749),題目:CTCF在單純皰疹病毒Ⅱ型潛伏復(fù)發(fā)中的作用研究~~
【分類號(hào)】：R373.11

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