本文關(guān)鍵詞：人巨細(xì)胞病毒蛋白pUL23影響宿主蛋白R(shí)ACK1與STAT1間的相互作用　出處：《暨南大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：人巨細(xì)胞病毒（Human cytomegalovirus HCMV）屬皰疹病毒科β亞屬（β皰疹病毒）,在人群中的感染率十分普遍。HCMV像其它β皰疹病毒一樣，不能被宿主體內(nèi)的免疫系統(tǒng)完全清除，它會(huì)以一種較低的病毒水平保持對(duì)宿主的持續(xù)感染或者是以一種非活性狀態(tài)持續(xù)潛伏于宿主體內(nèi)，因而對(duì)于免疫功能正常的人，HCMV可長(zhǎng)期潛伏而不致病。但是當(dāng)宿主免疫功能低下時(shí)，HCMV則可大量繁殖，進(jìn)而引起嚴(yán)重的致死并發(fā)癥。隨著目前多個(gè)HCMV病毒株完整基因組核酸序列的分析完成，HCMV基因組結(jié)構(gòu)已基本被闡明。通過(guò)對(duì)病毒臨床株基因的分析和缺失突變等發(fā)現(xiàn)，在200多個(gè)開(kāi)放閱讀框中（Open Reading Frame，ORF），僅有45到57個(gè)ORFs為病毒在人成纖維細(xì)胞中繁殖所必要，余下的都為非必需基因。研究表明，部分病毒蛋白參與了協(xié)調(diào)與宿主間細(xì)胞關(guān)系，，以達(dá)到自身與宿主細(xì)胞長(zhǎng)期共生的目的。 UL23基因是HCMV US22基因家族的成員，它能編碼一個(gè)大小為33kD的病毒皮層蛋白pUL23,聚集在細(xì)胞質(zhì)中的核周邊區(qū)域。然而對(duì)于pUL23蛋白的功能目前了解甚少。為了進(jìn)一步闡明pUL23的潛在功能，本實(shí)驗(yàn)室前期工作利用酵母雙雜交方法，從人胚腎cDNA文庫(kù)中篩選到多個(gè)與pUL23相互作用的宿主蛋白。RACK1[Receptor for activated C kinase1]就是其中之一。RACK1是蛋白激酶C的受體蛋白，因其可以和多種類(lèi)型的蛋白分子相結(jié)合，從而被普遍地認(rèn)為是一種多功能腳手架蛋白。本課題利用回復(fù)性酵母雙雜交、免疫共沉淀等實(shí)驗(yàn)方法進(jìn)一步確認(rèn)了pUL23與RACK1間的相互作用,且pUL23和RACK1共定位于細(xì)胞質(zhì)中。 研究發(fā)現(xiàn)RACK1能夠與非磷酸化的STAT1結(jié)合，并作為一個(gè)腳手架蛋白發(fā)揮了招募STAT1到干擾素受體上的功能。而STAT1與干擾素受體的預(yù)先結(jié)合是STAT1活化和干擾素信號(hào)途徑傳導(dǎo)必不可少的一步，這說(shuō)明RACK1與STAT1的結(jié)合對(duì)STAT1的活化和干擾素信號(hào)途徑的傳導(dǎo)是至關(guān)重要的。因此本文利用GSTpull-down實(shí)驗(yàn)檢測(cè)了pUL23與RACK1的結(jié)合對(duì)RACK1-STAT1相互結(jié)合的影響，實(shí)驗(yàn)結(jié)果表明pUL23減弱了RACK1與STAT1間的親和力，這說(shuō)明病毒蛋白pUL23能夠更牢固的“抓住”RACK1蛋白，進(jìn)而導(dǎo)致RACK1、STAT1或許還有干擾素受體在內(nèi)的復(fù)合物的解離。由文獻(xiàn)可知，RACK1與STAT1結(jié)合的減弱會(huì)直接導(dǎo)致STAT1磷酸化水平的下降和干擾素抗病毒信號(hào)途徑傳導(dǎo)的受阻。因此本文的研究結(jié)果提高了pUL23在干擾素信號(hào)途徑中發(fā)揮調(diào)節(jié)作用的可能性，并為今后pUL23蛋白功能的研究提供了諸多有意義的線(xiàn)索。
[Abstract]:Human cytomegalovirus (Human cytomegalovirus HCMV) is a beta herpesvirus subgenus (beta herpesvirus). The infection rate in population is very common.HCMV like other beta herpes virus, can not be completely removed the host immune system in the body, it will be in a low level of virus infection on the host to keep or in a non active state persistent in the host body, and for people with normal immune function, HCMV can lurk without disease. But when the host immune function is low, HCMV can multiply, causing serious complications. With the death of nucleic acid sequence analysis of multiple strains of HCMV complete genome completion HCMV, the genome structure has been elucidated. Through the analysis of clinical strains of virus and lack of gene mutations found in the more than 200 open reading frame (Open Reading Frame, ORF), only 45 to 57 ORFs is necessary for virus propagation in human fibroblasts, and the rest are non essential genes. Studies show that some viral proteins are involved in coordinating cell relationship with host cells, so as to achieve long-term purpose of their own host cells.
UL23 gene is a member of the HCMV US22 gene family encoding, it can a 33kD virus tegument protein pUL23, nuclear surrounding areas gathered in the cytoplasm. However, the function of pUL23 protein is currently poorly understood. In order to further elucidate the potential function of pUL23, the previous work in our laboratory using yeast two hybrid screening methods, from the people embryo kidney cDNA library to multiple interactions with the pUL23.RACK1[Receptor for activated C kinase1] host protein is one of the.RACK1 receptor protein protein kinase C, due to its many types of protein molecules and the combination, which was widely believed to be a multifunctional protein scaffold. The recovery of yeast two hybrid and Co immunoprecipitation experiments further confirmed the interaction between RACK1 and pUL23, and pUL23 and RACK1 were localized in the cytoplasm.
The research found that RACK1 and non phosphorylated STAT1 binding, and as a scaffolding protein play on the recruitment of STAT1 to interferon receptor function. STAT1 and interferon receptor binding is essential for pre STAT1 activation and interferon signal transduction step, which indicates that the combination of RACK1 and STAT1 on activation of STAT1 and interferon signaling the way of transmission is very important. So we use GSTpull-down test to detect effects of combination of pUL23 and RACK1 on RACK1-STAT1 with each other, the experimental results show that pUL23 can decrease the affinity between STAT1 and RACK1, suggesting that the virus protein pUL23 can be more firmly hold RACK1 protein, leading to RACK1, STAT1 may have complex dissociation compound interferon receptor including. By literature, weaken the RACK1 combined with STAT1 will directly lead to the phosphorylation level of STAT1 decreased and interference Therefore, the results of this study improve the possibility of pUL23 regulating the function of IFN signaling pathway, and provide many meaningful clues for the research of pUL23 protein function in the future.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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