HIV-1 整合酶3’端加工抑制剂筛选方法的建立及应用
文献类型：学位论文
| 作者 | 陆翠林 |
| 学位类别 | 硕士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 关键词 | HIV-1 整合酶 3’端加工 荧光共振能量转移技术 抑制剂 |
| 其他题名 | Establishment of a Method for Screening HIV-1 Integrase 3’-processing Inhibitor and its Application |
| 学位专业 | 生物工程 |
| 中文摘要 | 整合酶(Integrase，IN)是HIV感染和复制过程中的关键酶。它通过3’端加工和链转移2个功能，将病毒的cDNA整合到宿主细胞基因组中。由于宿主细胞中没有该酶的类似物，所以整合酶成为抗HIV药物筛选的理想靶点。 目前上市的3个整合酶抑制剂均为链转移抑制剂，整合酶3’端加工抑制剂报道较少，用于筛选整合酶3’端加工抑制剂的方法也存在一定的局限性，因此建立简单、快速的整合酶3’端加工抑制剂筛选方法对于发现新的整合酶抑制剂具有重要意义。本文利用荧光共振能量转移技术建立整合酶3’端加工抑制剂的筛选方法。先模拟病毒LTR合成底物DNA，并在底物DNA的5’端标记荧光基团FAM，3’端标记淬灭基团DABCYL，利用DNaseⅠ切割底物DNA确定底物检测的激发波长为495nm，发射波长为525nm，在该检测波长下，对缓冲液成分、底物浓度、酶浓度、金属离子浓度、反应时间、NaCl浓度、DMSO浓度等影响整合酶活性的条件进行优化，最后确定使用缓冲液1、底物浓度为500nM、整合酶浓度为1μM、镁离子浓度为20mM时整合酶3’端加工活性最强，反应6小时后进行检测信噪比较佳。用阳性药物雷特格韦和杨梅黄素对建立的方法进行验证，发现雷特格韦和杨梅黄素均能有效抑制整合酶3’端加工活性，证明建立的方法可用于整合酶3’端加工抑制剂的筛选。本文先对待检化合物进行细胞水平的抗HIV活性检测，对筛选到的有较强抗HIV活性的化合物再确定其是否对整合酶3’端加工有抑制作用，该方法可以降低假阳性现象。细胞水平筛选到的对HIV有抑制作用的25个化合物中经检测发现有10个化合物具有抑制整合酶3’端加工活性的作用，其中化合物6、7、9、10、14能明显的抑制整合酶3’端加工活性，其对整合酶3’端加工抑制的IC50值分别为 41.55μg/ml、56.69μg/ml、61.05μg/ml、16.58μg/ml、218.99μg/ml。综上所述，本研究成功的建立了体外整合酶3’端加工抑制剂筛选方法。采用建立的方法我们筛选到10个整合酶3’端加工抑制剂。整合酶3’端加工抑制剂筛选方法的建立不仅拓宽了HIV整合酶抑制剂的筛选范围，而且能更好的帮助我们了解整合酶的作用机理。 |
| 英文摘要 | Integrase (IN) of the human immunodeficiency virus 1 (HIV-1) is a key enzyme for HIV-1 infection and replication, which is responsible for integration of the viral cDNA into the host cell genome by 3’-processing and strand transfer activity. There is not analogy integrase in host cell. IN is an attractive target for screening anti-HIV-1 drugs.Now, three integrase inhibitors on the market are integrase strand transfer inhibitor. There are little integrase 3’-processing inhibitor in research reported. And the establishment assays for screening integrase 3’-processing inhibitor in vitro have some limintation. Hence, establishing a simple, rapid method for screening HIV-1 integrase 3’-processing inhibitor is very important to discovery new integrase inhibitor.In this thesis, we use fluorescence resonance energy transfer technology to create an assay for screening integrase 3’-processing inhibitors. The steps are as follows: 1) Synthesis a substrate DNA which analogs viral LTR DNA, lable FAM fluorescent group in the 5’ end and quencher DABCYL in the 3’ end of the DNA. 2) The testing wavelength defined by DNaseⅠare 495nm excitation and 525nm emission. 3) Under detection wavelength, buffer composition, substrate concentration, enzyme concentration, metal ion concentration, reaction time, NaCl concentration, DMSO concentration which affect integrase activity are tested to optimize reaction conditions. Finally, integrase 3’-processing optimize reaction conditions are buffer1, 500nM substrate, 1μM integrase, 20mM magnesium ion. Signal to Noise Ratio is preferably after 6 hours of the reaction. Positive drug raltegravir and myricetin can effectively inhibit integrase 3’-processing activity using this assy.So the established method can be used to screen integrase 3 '-processing inhibitor.The compounds are detected anti-HIV activity in cell level first in order to reduce false positive. Then, the compounds which have strong anti-HIV activity were tested to anti integrase 3’-processing.There are 25 compounds have robust anti-HIV activity in cell level and 10 samples of the 25 compounds can inhibit integrase 3’-processing. In the 10 compounds, compound 6, compound 7, compound 9, compound 10 and compound 14 strongly inhibit integrase 3’-processing activity, the IC50 values of these samples were 41.55μg/ml , 56.69μg/ml, 61.05μg/ml, 16.58ug/ml, 218.99μg/ml respectively.In summary, we successfully established a method to screening HIV integrase 3’-processing inhibitor. Using this method, we have screened 10 HIV integrase 3’-processing inhibitors which also have strong anti-HIV-1 activity in cell level. The method we are established not only broadens the screening range of HIV integrase inhibitor, but also helps us understand the catalytic mechanism of integrase. |
| 语种 | 中文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10203]  |
| 专题 | 昆明动物研究所_分子免疫药理学 |
| 推荐引用方式 GB/T 7714 | 陆翠林. HIV-1 整合酶3’端加工抑制剂筛选方法的建立及应用[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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