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  本文選題：HIV-1整合酶 + 慢病毒載體　； 參考：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：HIV-1整合酶(HIV-1 integrase, HTV-1 IN)是HIV-1慢病毒載體中的關(guān)鍵因子,在HIV-1基因整合入宿主細(xì)胞基因組過程中起重要作用。除整合病毒基因組功能外,HIV-1 IN還參與病毒生命周期的很多重要過程,如影響病毒粒子形態(tài)、病毒DNA合成和核內(nèi)運(yùn)輸?shù)�。本研究�?我們用酵母雙雜交系統(tǒng)發(fā)現(xiàn)HIV-1 IN中的四肽156KELK159在HIV-1 IN與Daxx結(jié)合中發(fā)揮重要作用,缺失該四肽的HIV-1 IN無法與Daxx結(jié)合。為進(jìn)一步探索156KELK159在HIV-1慢病毒載體基因整合及表達(dá)中的作用,我們利用三質(zhì)粒包裝系統(tǒng)分別包裝出含野生型HIV-1 IN及含缺失156KELK159的HIV-1 IN兩種均以EGFP為報(bào)告基因的慢病毒載體。使用p24病毒滴度檢測試劑盒檢測病毒滴度,同時抽取病毒RNA,利用實(shí)時定量PCR檢測病毒液中的RNA拷貝數(shù),結(jié)果發(fā)現(xiàn)相同p24含量的兩種病毒中的RNA拷貝數(shù)也相同,故156KELK159對病毒載體的包裝沒有影響。進(jìn)一步研究發(fā)現(xiàn)156KELK159在HIV-1慢病毒載體基因的整合過程中發(fā)揮重要作用。兩種病毒分別感染293T細(xì)胞后,156KELK159的缺失使被感染細(xì)胞中表達(dá)報(bào)告基因的細(xì)胞比例明顯降低,但進(jìn)入細(xì)胞內(nèi)及進(jìn)入細(xì)胞核內(nèi)的DNA水平未發(fā)生變化。逆轉(zhuǎn)錄實(shí)時定量PCR及FACS被用于檢測感染維胞中報(bào)告基因EGFP的表達(dá)水平,結(jié)果發(fā)現(xiàn)156KELK159并不影響HIV-1慢病毒載體的報(bào)告基因表達(dá)。本研究結(jié)果為進(jìn)一步揭示HIV-1慢病毒載體基因整合的調(diào)控機(jī)制提供了一定理論依據(jù)。
[Abstract]:HIV-1 integrase HIV-1 integrase (HTV-1 IN) is a key factor in HIV-1 lentivirus vector and plays an important role in the integration of HIV-1 gene into host cell genome. In addition to integrating viral genome functions, HIV-1 IN also participates in many important processes of virus life cycle, such as affecting virus particle morphology, virus DNA synthesis and nuclear transport. In this study, we used yeast two-hybrid system to find that tetrapeptide 156KELK159 in HIV-1 IN plays an important role in the binding of HIV-1 IN to Daxx, and HIV-1 IN without this tetrapeptide can not bind to Daxx. In order to further explore the role of 156KELK159 in the gene integration and expression of HIV-1 lentivirus vector, we used three plasmids packaging system to package lentivirus vectors containing wild type HIV-1 IN and HIV-1 IN containing missing 156KELK159, respectively. EGFP was used as the reporter gene of lentivirus vectors. The virus titer was detected by using p24 virus titer detection kit and virus RNAs were extracted at the same time. The RNA copy number in the virus solution was detected by real-time quantitative PCR. The results showed that the RNA copy number of the two viruses with the same p24 content was the same. So 156KELK159 has no effect on the packaging of virus vector. Further studies have found that 156KELK159 plays an important role in the integration of HIV-1 lentivirus vector genes. After the two viruses were infected with 293T cells respectively, the deletion of 156KELK159 significantly reduced the proportion of cells expressing reporter gene in infected cells, but the level of DNA entering into cell and nucleus did not change. Reverse transcription real-time PCR and FACS were used to detect the expression of reporter gene EGFP in infected cells. The results showed that 156KELK159 did not affect the expression of reporter gene in HIV-1 lentivirus vector. The results provide a theoretical basis for further revealing the regulation mechanism of HIV-1 lentivirus vector gene integration.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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相關(guān)期刊論文 前2條

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本文編號：1881913