【摘要】： 乙型肝炎病毒(HBV)基因組中的X基因及其編碼產(chǎn)物X蛋白(hepatitis B virus X protein, HBx)在HBV的復(fù)制中起著重要的調(diào)節(jié)作用。HBx參與多條細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路,對(duì)細(xì)胞的增殖,凋亡發(fā)揮重要的調(diào)節(jié)作用。它主要與細(xì)胞信號(hào)通路中一些關(guān)鍵性的調(diào)節(jié)蛋白質(zhì)協(xié)同完成對(duì)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路的調(diào)節(jié)作用。這些調(diào)控作用包括激活宿主細(xì)胞和病毒自身基因的轉(zhuǎn)錄、調(diào)控凋亡、抑制細(xì)胞中受損DNA的外切修復(fù)反應(yīng)以及激活細(xì)胞中的信號(hào)轉(zhuǎn)導(dǎo)通路如有絲分裂原(MAPK)、JAK、STAT的級(jí)聯(lián)反應(yīng)等。但是在HBx參與眾多信號(hào)轉(zhuǎn)導(dǎo)通路中,我們關(guān)注HBx激活MAPK信號(hào)轉(zhuǎn)導(dǎo)通路,該通路是一條高度保守的模塊,與細(xì)胞各種功能有關(guān),包括細(xì)胞增殖,分化和遷移等,并且在腫瘤形成和遷移過(guò)程中發(fā)揮巨大的調(diào)節(jié)作用。但是目前并未發(fā)現(xiàn)HBx直接作用與MAPK信號(hào)中的蛋白酶受體,其激活的潛在分子機(jī)制尚未闡明。通過(guò)本課題的研究來(lái)初步闡明HBx編碼的蛋白質(zhì)在肝臟中是通過(guò)調(diào)節(jié)MAPK的抑制受體---SPRY1基因編碼的蛋白質(zhì)來(lái)激活MAPK信號(hào)通路。為了闡明該機(jī)制,本課題采取了一系列實(shí)驗(yàn)研究：(1)構(gòu)建Flag-HBx、Myc-SPRY1 Myc-SPRY1-53-Mut真核表達(dá)載體；(2)確定Flag-HBx和Myc-SPRY1蛋白質(zhì)之間是否存在相互作用；(3)探索Flag-HBx編碼的蛋白質(zhì)與Myc-SPRY1編碼蛋白質(zhì)相互作用結(jié)構(gòu)域的研究；(4)Myc-SPRY1編碼蛋白質(zhì)是否影響Flag-HBx在哺乳動(dòng)物HEK293細(xì)胞中的蛋白質(zhì)表達(dá)水平；(5)AP-1熒光素酶報(bào)告基因?qū)嶒?yàn)研究Flag-HBx和Myc-SPRY1編碼的蛋白質(zhì)對(duì)AP-1轉(zhuǎn)錄因子的影響；(6)研究Flag-HBx和Myc-SPRY1編碼的蛋白質(zhì)對(duì)HEK293細(xì)胞凋亡、增殖的功能影響。所得結(jié)論如下： 1.成功構(gòu)建Flag-HBx、Myc-SPRY1、Myc-SPRY1-53-Mut,真核表達(dá)載體； 2.通過(guò)免疫共沉淀(Co-IP)和Pull-down實(shí)驗(yàn)確定了Flag-HBx和Myc-SPRY1在哺乳動(dòng)物HEK293細(xì)胞體內(nèi)外存在相互作用； 3.通過(guò)免疫共沉淀實(shí)驗(yàn)表明SPRY1第53位酪氨酸突變體并不影響它們之間的相互作用,但是AP-1熒光素酶報(bào)告基因?qū)嶒?yàn)表明SPRY1第53位酪氨酸突變體影響它與HBx之間對(duì)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路的激活作用,因此我們推測(cè)它們相互作用區(qū)域是包括這一點(diǎn)的一段序列； 4. Myc-SPRY1編碼蛋白質(zhì)影響Flag-HBx編碼的蛋白質(zhì)在哺乳動(dòng)物HEK293細(xì)胞的表達(dá)水平,并且Flag-HBx編碼的蛋白質(zhì)水平隨著Myc-SPRY1編碼蛋白質(zhì)表達(dá)水平的增加而增加； 5. Flag-HBx協(xié)同Myc-SPRY1對(duì)核轉(zhuǎn)錄因子AP-1-luc的轉(zhuǎn)錄活性具有激活效應(yīng)； 6.通過(guò)細(xì)胞凋亡實(shí)驗(yàn)和細(xì)胞增殖實(shí)驗(yàn)發(fā)現(xiàn),Flag-HBx促進(jìn)HEK293細(xì)胞的增殖,抑制HEK293細(xì)胞的凋亡；并且發(fā)現(xiàn)當(dāng)共轉(zhuǎn)染Myc-SPRY1和Flag-HBx到HEK293細(xì)胞中時(shí),Myc-SPRY1增加了Flag-HBx對(duì)HEK293細(xì)胞的抗凋亡作用,并且Myc-SPRY1上調(diào)了Flag-HBx對(duì)HEK293細(xì)胞的增殖水平的促進(jìn)作用。
[Abstract]:X gene and X protein (hepatitis B virus X protein, HBx) in the genome of hepatitis B virus (HBV) play an important role in the replication of HBV. HBX participates in many cell signal transduction pathways and proliferates cells. Apoptosis plays an important regulatory role. It mainly works with some key regulatory proteins in cell signaling pathway to regulate cell signal transduction pathway. These effects include activation of transcription of host cells and virus genes, regulation of apoptosis, inhibition of extracellular repair of damaged DNA in cells and activation of signal transduction pathways in cells, such as the cascade of mitogen (MAPK) (MAPK) JAKTSTAT, and so on. However, among the many signal transduction pathways in which HBx is involved, we focus on the activation of MAPK signal transduction pathway by HBx, which is a highly conserved module related to various cellular functions, including cell proliferation, differentiation and migration. And play a huge regulatory role in tumor formation and migration. However, no direct interaction of HBx with protease receptor in MAPK signal has been found, and the underlying molecular mechanism of its activation has not been elucidated. In this study, we preliminarily demonstrated that the HBx encoded protein activates the MAPK signaling pathway by regulating the protein encoded by the suppressor SPRY1 gene of MAPK in the liver. In order to elucidate the mechanism, a series of experimental studies were carried out: (1) constructing the eukaryotic expression vector of Flag-HBxSPRY1 Myc-SPRY1-53-Mut, (2) determining the interaction between Flag-HBx and Myc-SPRY1 protein; (3) to explore the interaction domain between Flag-HBx encoded proteins and Myc-SPRY1 encoded proteins, (4) whether Myc-SPRY1 encoded proteins affect the expression of Flag-HBx in mammalian HEK293 cells; (5) the effects of Flag-HBx and Myc-SPRY1 encoded proteins on AP-1 transcription factors were studied by AP-1 luciferase reporter gene experiment. (6) the effects of Flag-HBx and Myc-SPRY1 encoded proteins on the apoptosis and proliferation of HEK293 cells were studied. The conclusions are as follows: 1. The eukaryotic expression vector of Flag-HBxCPRY1, Myc-SPRY1-53-Mut. was successfully constructed. The interaction between Flag-HBx and Myc-SPRY1 in mammalian HEK293 cells in vivo and in vitro was confirmed by immunoprecipitation (Co-IP) and Pull-down assay. The results of immunoprecipitation showed that tyrosine mutants at position 53 of SPRY1 did not affect the interaction between them. However, the AP-1 luciferase reporter gene experiment showed that tyrosine mutants at position 53 of SPRY1 affect the activation of signal transduction pathway between SPRY1 and HBx, so we speculate that their interaction region is a sequence including this. 4. Myc-SPRY1 encoded protein affects the expression level of Flag-HBx encoded protein in mammalian HEK293 cells, and Flag-HBx protein level increases with the increase of Myc-SPRY1 protein expression level. 5. Flag-HBx combined with Myc-SPRY1 could activate the transcription activity of nuclear transcription factor AP-1-luc. It was found that Flag-HBx promoted the proliferation of HEK293 cells and inhibited the apoptosis of HEK293 cells, and that Myc-SPRY1 increased the anti-apoptotic effect of Flag-HBx on HEK293 cells when co-transfected with Myc-SPRY1 and Flag-HBx into HEK293 cells. Myc-SPRY1 upregulated the promotion of Flag-HBx on the proliferation of HEK293 cells.
【學(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373

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本文編號(hào)：2181272