HIV-1整合酶的四肽156KELK159在慢病毒载体基因整合过程中的作用研究

发布时间：2018-05-13 05:38

本文选题：HIV-1整合酶 + 慢病毒载体　；参考：《复旦大学》2011年硕士论文

【摘要】：HIV-1整合酶(HIV-1 integrase, HTV-1 IN)是HIV-1慢病毒载体中的关键因子,在HIV-1基因整合入宿主细胞基因组过程中起重要作用。除整合病毒基因组功能外,HIV-1 IN还参与病毒生命周期的很多重要过程,如影响病毒粒子形态、病毒DNA合成和核内运输等。本研究中,我们用酵母双杂交系统发现HIV-1 IN中的四肽156KELK159在HIV-1 IN与Daxx结合中发挥重要作用,缺失该四肽的HIV-1 IN无法与Daxx结合。为进一步探索156KELK159在HIV-1慢病毒载体基因整合及表达中的作用,我们利用三质粒包装系统分别包装出含野生型HIV-1 IN及含缺失156KELK159的HIV-1 IN两种均以EGFP为报告基因的慢病毒载体。使用p24病毒滴度检测试剂盒检测病毒滴度,同时抽取病毒RNA,利用实时定量PCR检测病毒液中的RNA拷贝数,结果发现相同p24含量的两种病毒中的RNA拷贝数也相同,故156KELK159对病毒载体的包装没有影响。进一步研究发现156KELK159在HIV-1慢病毒载体基因的整合过程中发挥重要作用。两种病毒分别感染293T细胞后,156KELK159的缺失使被感染细胞中表达报告基因的细胞比例明显降低,但进入细胞内及进入细胞核内的DNA水平未发生变化。逆转录实时定量PCR及FACS被用于检测感染维胞中报告基因EGFP的表达水平,结果发现156KELK159并不影响HIV-1慢病毒载体的报告基因表达。本研究结果为进一步揭示HIV-1慢病毒载体基因整合的调控机制提供了一定理论依据。
[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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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