本文關(guān)鍵詞：人細(xì)小病毒B19類(lèi)染色質(zhì)結(jié)構(gòu)調(diào)控基因組復(fù)制及RNA加工　出處：《中國(guó)科學(xué)院武漢病毒研究所》2017年博士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：人類(lèi)細(xì)小病毒B19(B19V)屬于細(xì)小病毒科的嗜紅細(xì)胞屬,基因組為單鏈DNA,全長(zhǎng)5.6kb。B19V和人博卡病毒是現(xiàn)今可以感染人類(lèi)的僅有的兩種細(xì)小病毒科的病毒。B19V感染主要引起傳染性紅斑、急性再生障礙性貧血、關(guān)節(jié)炎等,感染孕婦后可導(dǎo)致胎兒水腫,流產(chǎn)或者死胎。B19V基因組內(nèi)有兩個(gè)polyA信號(hào),位于3'端的稱(chēng)為(pA)d,位于第二個(gè)內(nèi)含子內(nèi)的稱(chēng)為(pA)p。(pA)p直接抑制病毒轉(zhuǎn)錄出全長(zhǎng)pre-mRNA,在轉(zhuǎn)錄后水平與第二個(gè)內(nèi)含子的剪切相互競(jìng)爭(zhēng)抑制病毒結(jié)構(gòu)蛋白mRNA的生成,而病毒的復(fù)制利于啟動(dòng)子通讀過(guò)(pA)p位點(diǎn),使用(pA)d位點(diǎn)。病毒基因組的復(fù)制和第二個(gè)內(nèi)含子的剪切都影響(pA)p的使用,但是復(fù)制如何影響(pA)p的使用還不清楚。細(xì)小病毒MVM和腺相關(guān)病毒AAV感染敏感細(xì)胞后,可以和組蛋白結(jié)合,形成類(lèi)染色質(zhì)結(jié)構(gòu),但是類(lèi)染色質(zhì)結(jié)構(gòu)有哪些功能還沒(méi)有相關(guān)報(bào)道。因此,本研究主要目的是探討B(tài)19V基因組是否可以在細(xì)胞內(nèi)形成類(lèi)染色質(zhì)結(jié)構(gòu),研究B19V基因組甲基化修飾以及類(lèi)染色質(zhì)結(jié)構(gòu)對(duì)基因復(fù)制和RNA加工的影響,進(jìn)一步揭示基因組復(fù)制影響(pA)p使用的分子機(jī)制。為了研究B19V基因組DNA是否形成類(lèi)染色質(zhì)結(jié)構(gòu),轉(zhuǎn)染B19V DNA到HEK293T細(xì)胞內(nèi),分別在12、24、36和48小時(shí)提取細(xì)胞核,微球菌核酸酶消化后用苯酚-氯仿抽提,同時(shí)在這四個(gè)時(shí)間段提取Hirt DNA,Southern blot檢測(cè)。結(jié)果證實(shí)B19V基因組DNA可以在細(xì)胞內(nèi)形成類(lèi)染色質(zhì)結(jié)構(gòu),且類(lèi)染色質(zhì)結(jié)構(gòu)的形成早于復(fù)制。為了研究病毒染色質(zhì)的修飾是否影響病毒的復(fù)制、轉(zhuǎn)錄或轉(zhuǎn)錄后加工,我們應(yīng)用甲基轉(zhuǎn)移酶抑制劑5-Aza-2’-deoxycytidine(DAC)處理轉(zhuǎn)染B19V基因組DNA后的HEK293T細(xì)胞,48小時(shí)后提取小分子量DNA(Hirt DNA),利用DpnI消化,用甲基化檢測(cè)試劑盒處理后進(jìn)行PCR擴(kuò)增反應(yīng),通過(guò)測(cè)序我們發(fā)現(xiàn),DAC處理后基因組上特定位點(diǎn)的甲基化修飾被抑制,表明在B19V基因組內(nèi)存在甲基化修飾位點(diǎn)。用終濃度為20μM的DAC處理轉(zhuǎn)染后的293T細(xì)胞,48h后分別提取細(xì)胞核、Hirt DNA和總RNA,并用Southern blot和RNA酶保護(hù)實(shí)驗(yàn)進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn),與DMSO處理組和未處理組比較,DAC明顯抑制B19V類(lèi)染色質(zhì)結(jié)構(gòu)的形成及基因組的復(fù)制。RPA實(shí)驗(yàn)證實(shí)DAC抑制第二個(gè)內(nèi)含子的剪切,促進(jìn)(pA)p的使用。為了研究B19V基因組復(fù)制影響(pA)p使用的具體機(jī)制,分別利用IRES2-eGFP和pBluescript KS II載體構(gòu)建了基因組5’和3’端一半基因的復(fù)制型和非復(fù)制型克隆,并在此基礎(chǔ)上獲得了敲除D1和D2剪切位點(diǎn)的克隆,轉(zhuǎn)染HEK293T細(xì)胞,48小時(shí)后提取總RNA,RNA酶保護(hù)實(shí)驗(yàn)進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn)B19V基因組的復(fù)制主要通過(guò)影響第二個(gè)內(nèi)含子的剪切影響(pA)p的使用。本研究證實(shí)B19V基因組上存在甲基化修飾,DAC可以使基因組去甲基化,抑制類(lèi)染色質(zhì)結(jié)構(gòu)的形成和復(fù)制,導(dǎo)致基因組第二個(gè)內(nèi)含子的剪切被抑制,進(jìn)而影響(pA)p的使用。本研究首次報(bào)道了B19V基因組甲基化修飾和類(lèi)染色質(zhì)結(jié)構(gòu)影響基因組的復(fù)制和RNA轉(zhuǎn)錄后加工,揭示了B19V基因組中央poly位點(diǎn)選擇性使用的分子調(diào)控機(jī)制,為B19V的防治提供了理論依據(jù)。
[Abstract]:Human parvovirus B19 (B19V) belongs to the Parvoviridae of eosinophilic cells, single stranded DNA genome, full-length 5.6kb.B19V and Boka virus can infect humans is now only two minute virus of.B19V infection mainly caused by infectious erythema, acute aplastic anemia, arthritis, after infection of pregnant women lead to fetal edema, abortion or stillbirth within the.B19V genome with two polyA signal in 3'terminal called D (pA), located in the second intron called (pA) P. (pA) P directly inhibit viral transcription of full-length pre-mRNA, competing suppression of virus structural protein mRNA in shear transcription after the level and second introns, and the replication of the virus to read through the promoter (pA) P locus, using (pA) d site. The shear replication of the virus genome and second introns are affected (pA) the use of P, but how to copy. Ring (pA) the use of P is not clear. Parvovirus MVM and adeno-associated virus AAV infection sensitive cells, and can be combined with histones, chromatin structure formation, but the type of chromatin structure which function has not been reported yet. Therefore, the main purpose of this study is to investigate whether B19V gene group can be formed the class structure of chromatin in the cells, the study of B19V genome methylation and influence of chromatin structure on gene duplication and RNA processing, to further reveal the influence of genome replication (pA) molecular mechanism used by P. In order to study the B19V genomic DNA formation of chromatin structure, transfection of B19V DNA into HEK293T cells were extracted in 48 hours and 12,24,36 nuclei, micrococcal nuclease digestion by phenol chloroform extraction, DNA extraction and Hirt in the four time, Southern blot detection. The results demonstrated that B19V genome DNA can be in the cell In the formation of chromatin structure, formation and chromatin structure in early replication. In order to study whether the virus modified chromatin influence viral replication, transcription or post transcription processing, we used the methyltransferase inhibitor 5-Aza-2 -deoxycytidine (DAC) transfected B19V gene group DNA after the HEK293T cells after 48 hours the extraction of low molecular weight DNA (Hirt DNA), using DpnI digestion treatment test kit with methyl PCR after amplification by sequencing, we found that after the treatment of DAC methylation specific sites on the genome was inhibited, showed that in the memory in the B19V genome methylation sites. With a final concentration of 20 M DAC after transfection of 293T cells after 48h nuclei were extracted, Hirt DNA and RNA, and were detected by Southern blot and RNA enzyme protection experiments. The results showed that DMSO treatment group and untreated group, DAC significantly Copy the.RPA experimental inhibition of B19V dyeing formation and genome structure of the matter confirmed that the shear DAC inhibition of the second introns (pA) to promote the use of P. In order to study the influence of B19V genome replication (pA) specific mechanism used by P, IRES2-eGFP and pBluescript respectively by KS II carrier to construct genomic 5 'and 3' at the end of half gene replicating and non replicating clones, and based on the cloning of knock except D1 and D2 splice sites, were transfected into HEK293T cells, 48 hours after the total RNA was extracted from experiment RNA enzyme protection were detected. The results showed that B19V genome replication mainly through the influence of shear second introns (pA the use of P). This study confirmed the presence of methylation of the B19V genome, DAC can make the genome demethylation and inhibition of chromatin structure formation and replication, leading to shear genomic second introns were inhibited, and Effect of P (pA). This is the first report of B19V genome methylation and chromatin structure of genome replication and RNA transcription, revealing the molecular mechanism of B19V genome poly central site selective use, provides a theoretical basis for the prevention and treatment of B19V.

【學(xué)位授予單位】：中國(guó)科學(xué)院武漢病毒研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q78;R373

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