發(fā)布時(shí)間：2018-04-13 09:53

  本文選題：乙型肝炎病毒 + RNA剪接　； 參考：《福建醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 乙型肝炎病毒基因組剪接變異體( spliced variants of hepatitis B virus genomes)是一大類基因部分缺失的亞基因組DNA,由乙型肝炎病毒(Hepatitis B virus, HBV)前基因組RNA (pregenomic RNA, pgRNA)經(jīng)剪接并逆轉(zhuǎn)錄產(chǎn)生[1]。在已經(jīng)分離到的HBV剪接變異體中,長(zhǎng)度約為2.2kb的剪接變異體占80%[2],此類變異體主要分為單剪接與雙剪接兩種類型。研究顯示這兩種HBV基因組剪接變異體編碼的剪接特異性新蛋白與病毒的持續(xù)性感染及致病性相關(guān)[3,4],但其確切的致病機(jī)制迄今不明。本研究通過(guò)人類基因表達(dá)譜芯片研究分析這兩種剪接特異性新蛋白對(duì)Huh7肝細(xì)胞基因表達(dá)的影響,探尋它們可能的致病機(jī)制。 本研究第一部分旨在構(gòu)建剪接特異性新蛋白真核表達(dá)載體,并證實(shí)其在肝細(xì)胞中的表達(dá)。為此將剪接特異性新蛋白編碼區(qū)克隆到真核表達(dá)載體pcDNA3.1/HisC ,獲得重組載體pcDNA3.1/HisC-TPds(雙剪接型)、pcDNA3.1/HisC-TPss(單剪接型),瞬時(shí)轉(zhuǎn)染Huh7肝細(xì)胞,以Anti-Xpress抗體進(jìn)行Western Blot檢測(cè)融合蛋白,證實(shí)剪接特異性新蛋白可在Huh7肝細(xì)胞中表達(dá)。 本研究第二部分以pcDNA3.1/HisC、pcDNA3.1/HisC-TPds、pcDNA3.1/HisC-TPss分別瞬時(shí)轉(zhuǎn)染Huh7肝細(xì)胞,提取轉(zhuǎn)染細(xì)胞總RNA,進(jìn)行人類基因表達(dá)譜芯片分析。同時(shí)以半定量RT-PCR驗(yàn)證芯片結(jié)果。芯片分析顯示:2.2kb HBV基因組剪接變異體剪接特異性新蛋白可能從干擾肝細(xì)胞骨架的重塑、細(xì)胞內(nèi)物質(zhì)代謝等方面引起肝細(xì)胞的增生、遷移及代謝異常,促進(jìn)腫瘤的發(fā)生。
[Abstract]:Spliced variants of hepatitis B virus genomes is a class of partially absent subgenomic DNAs, which is produced by splicing and reverse transcription of RNA pregenomic RNAs (pgRNAs) before hepatitis B virus (HBV).Among the isolated HBV splicing variants, about 80% of the splicing variants with length of 2.2kb are divided into two types: single splicing and double splicing.Studies have shown that the splicing specific proteins encoded by these two HBV genomic splicing variants are related to the persistent infection and pathogenicity of the virus.In this study, the effects of two splicing specific novel proteins on the gene expression of Huh7 hepatocytes were studied by using human gene expression microarray, and the possible pathogenetic mechanisms of these two novel splicing proteins were explored.The first part of this study was to construct a splicing specific eukaryotic expression vector and to confirm its expression in hepatocytes.Therefore, the splicing specific new protein coding region was cloned into eukaryotic expression vector pcDNA3.1/HisC, and the recombinant vector pcDNA3.1 / HisC-TPdswas obtained. The recombinant vector pcDNA3.1 / HisC-TPsss was transiently transfected into Huh7 hepatocytes, and the fusion protein was detected by Western Blot with Anti-Xpress antibody.It is confirmed that splicing specific new protein can be expressed in Huh7 hepatocytes.In the second part of the study, pcDNA3.1 / HisC-TPdsnpcDNA3.1 / HisC-TPSS pcDNA3.1 / HisC-TPss was used to transfect Huh7 hepatocytes, and the total RNAs were extracted and analyzed by human gene expression microarray.At the same time, semi-quantitative RT-PCR was used to verify the chip results.The microarray analysis showed that the splicing specific new protein of 2. 2kb HBV genomic splicing variant might cause hepatocyte proliferation, migration and abnormal metabolism, and promote the occurrence of tumor by interfering with the remodeling of hepatocyte cytoskeleton and intracellular substance metabolism.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 林旭,鄭大利,徐曉,林建銀;乙型肝炎病毒基因組剪接變異體結(jié)構(gòu)分析[J];中華傳染病雜志;2004年02期

，

本文編號(hào)：1743986