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  本文關鍵詞：人巨細胞病毒蛋白pUL23影響宿主蛋白RACK1與STAT1間的相互作用　出處：《暨南大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 人巨細胞病毒 pUL23 RACK1 STAT1 蛋白相互作用

【摘要】：人巨細胞病毒（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，不能被宿主體內的免疫系統(tǒng)完全清除，它會以一種較低的病毒水平保持對宿主的持續(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，因而對于免疫功能正常的人，HCMV可長期潛伏而不致病。但是當宿主免疫功能低下時，HCMV則可大量繁殖，進而引起嚴重的致死并發(fā)癥。隨著目前多個HCMV病毒株完整基因組核酸序列的分析完成，HCMV基因組結構已基本被闡明。通過對病毒臨床株基因的分析和缺失突變等發(fā)現，在200多個開放閱讀框中（Open Reading Frame，ORF），僅有45到57個ORFs為病毒在人成纖維細胞中繁殖所必要，余下的都為非必需基因。研究表明，部分病毒蛋白參與了協調與宿主間細胞關系，，以達到自身與宿主細胞長期共生的目的。 UL23基因是HCMV US22基因家族的成員，它能編碼一個大小為33kD的病毒皮層蛋白pUL23,聚集在細胞質中的核周邊區(qū)域。然而對于pUL23蛋白的功能目前了解甚少。為了進一步闡明pUL23的潛在功能，本實驗室前期工作利用酵母雙雜交方法，從人胚腎cDNA文庫中篩選到多個與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，因其可以和多種類型的蛋白分子相結合，從而被普遍地認為是一種多功能腳手架蛋白。本課題利用回復性酵母雙雜交、免疫共沉淀等實驗方法進一步確認了pUL23與RACK1間的相互作用,且pUL23和RACK1共定位于細胞質中。 研究發(fā)現RACK1能夠與非磷酸化的STAT1結合，并作為一個腳手架蛋白發(fā)揮了招募STAT1到干擾素受體上的功能。而STAT1與干擾素受體的預先結合是STAT1活化和干擾素信號途徑傳導必不可少的一步，這說明RACK1與STAT1的結合對STAT1的活化和干擾素信號途徑的傳導是至關重要的。因此本文利用GSTpull-down實驗檢測了pUL23與RACK1的結合對RACK1-STAT1相互結合的影響，實驗結果表明pUL23減弱了RACK1與STAT1間的親和力，這說明病毒蛋白pUL23能夠更牢固的“抓住”RACK1蛋白，進而導致RACK1、STAT1或許還有干擾素受體在內的復合物的解離。由文獻可知，RACK1與STAT1結合的減弱會直接導致STAT1磷酸化水平的下降和干擾素抗病毒信號途徑傳導的受阻。因此本文的研究結果提高了pUL23在干擾素信號途徑中發(fā)揮調節(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.

【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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