發(fā)布時(shí)間：2018-05-19 23:08

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

【摘要】： HBV基因組剪接變異體( spliced variants of hepatitis B virus genomes)是由HBV前基因組RNA (pregenomic RNA, pgRNA)經(jīng)剪接并逆轉(zhuǎn)錄產(chǎn)生的亞基因組DNA。長(zhǎng)度為2.2Kb的HBV剪接變異體占80%以上,分為雙剪接型和單剪接型,它們可編碼剪接特異性新蛋白并與病毒的持續(xù)性感染及致病性相關(guān)。本研究著眼于這兩種剪接變異體編碼的兩種不同的剪接特異性新蛋白,擬用酵母雙雜交的方法,尋找能夠與它們相互作用的細(xì)胞蛋白,從而探索它們的致病性。 本研究第一部分從臨床標(biāo)本中分離獲得雙剪接型與單剪接型2.2Kb乙型肝炎病毒基因組剪接變異體,在此基礎(chǔ)上構(gòu)建了雙剪接型剪接變異體特異性新基因(TPds基因)與單剪接型剪接變異體特異性新基因(TPss基因)的酵母雙雜交誘餌載體pGBKT7-TPds、pGBKT7-TPss。在酵母雙雜交實(shí)驗(yàn)過(guò)程中,發(fā)現(xiàn)TPds具有反式激活GAL4反應(yīng)元件的能力;而TPss則沒(méi)有這一功能。 本研究的第二部分旨在深入探討TPds的反式激活作用。利用分段克隆的方法,確定了TPds蛋白反式激活的主要功能區(qū)域位于其中部的28個(gè)氨基酸,此28個(gè)氨基酸編碼框符合preS1第30-57位氨基酸,從剪接體的角度闡明了具有致病意義的preS1相關(guān)蛋白的第二種來(lái)源。進(jìn)一步構(gòu)建TPds蛋白的哺乳動(dòng)物細(xì)胞表達(dá)載體pcDNA3.1/HisC-TPds,證實(shí)了該蛋白在肝細(xì)胞中對(duì)CMV即刻早期啟動(dòng)子、SV40啟動(dòng)子/增強(qiáng)子也具有反式激活作用,同時(shí)還證實(shí)了該蛋白對(duì)HBV自身啟動(dòng)子/增強(qiáng)子也具有刺激作用。 本研究的第三部分利用酵母雙雜交系統(tǒng)篩查肝細(xì)胞中與TPss蛋白相互作用的細(xì)胞蛋白,獲得四種候選蛋白:組織蛋白酶B、微粒體環(huán)氧化物水解酶、組織蛋白酶D、纖維蛋白原γ鏈;并通過(guò)哺乳動(dòng)物細(xì)胞雙雜交實(shí)驗(yàn)驗(yàn)證了它們與TPss蛋白在肝癌細(xì)胞Huh7中的相互作用;提示TPss可能影響肝細(xì)胞凋亡與腫瘤的侵襲轉(zhuǎn)移,并可能影響肝組織的解毒功能與凝血功能。
[Abstract]:HBV genomic splicing variant (spliced variants of hepatitis B virus genomes) is a subgenomic DNA produced by splicing and reverse transcription of pregenomic RNA pregenomic RNAs (pgRNAs). More than 80% of HBV splicing variants with length of 2.2Kb can be divided into double splicing type and single splicing type. They can encode splicing specific new protein and are related to persistent infection and pathogenicity of virus. This study aims at two different splicing specific proteins encoded by these two splicing variants. We intend to explore their pathogenicity by using yeast two-hybrid method to search for cellular proteins that can interact with them. In the first part of this study, double splicing and single splicing 2.2Kb HBV genomic splicing variants were isolated from clinical specimens. The yeast two-hybrid bait vector pGBKT7-TPSS was constructed based on the novel splicing variant specific gene (TPDS gene) and the single splice variant gene (pGBKT7-TPSS gene) of yeast two hybrid bait vector pGBKT7-TPKT7-TPss. based on the above results, the yeast two-hybrid decoy vector pGBKT7-TPKT7-TPssis was constructed. In yeast two-hybrid experiments, it was found that TPds had the ability to transactivate GAL4 reaction elements, but TPss did not. The second part of this study aims to explore the transactivation of TPds. By using the method of segmental cloning, 28 amino acids located in the middle of TPds protein were identified as the main functional region of transactivation. The 28 amino acid coding frames were in accordance with the 30-57 amino acids of preS1. The second source of preS1 associated proteins with pathogenicity was elucidated from splicing point of view. Further construction of mammalian expression vector pcDNA3.1 / HisC-TPdsof TPds protein confirmed that the protein could also transactivate the CMV immediate early promoter SV40 promoter / enhancer in hepatocytes. It is also confirmed that the protein also stimulates the promoter / enhancer of HBV itself. In the third part of this study, yeast two-hybrid system was used to screen cellular proteins interacting with TPss protein in hepatocytes. Four candidate proteins were obtained: cathepsin B, microsomal epoxide hydrolase, cathepsin D, fibrinogen 緯 chain. The interaction between TPss and TPss protein in Huh7 cells was verified by two-hybrid assay of mammalian cells, suggesting that TPss may affect hepatocyte apoptosis and tumor invasion and metastasis, and may affect the detoxification function and coagulation function of liver tissue.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R373.21

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本文編號(hào)：1912089