HIV-1复制中间产物检测方法的建立及在抗HIV药物研究中的初步应用
文献类型：学位论文
| 作者 | 陈欢 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郑永唐 |
| 关键词 | HIV-1 中间产物 qPCR 逆转录 整合 抗HIV药物 |
| 其他题名 | Establishment of Methods for Analysing Intermediate Products of HIV-1 Replication and Primary Application in Anti-HIV Drugs Study |
| 学位专业 | 工程硕士专业学位 |
| 中文摘要 | 由于缺乏有效的疫苗预防HIV感染，发现新的有效且经济的抗HIV药物，是艾滋病研究领域的当务之急。美国FDA 和中国SFDA关于抗HIV药物的临床前药效学评价指导原则中都明确指出，在抗HIV药物研究中，不仅要对抗HIV药物的抗病毒活性进行研究，还要对抗HIV药物的作用机制进行研究，为临床治疗方案和耐药性研究提供理论依据。 HIV-1复制过程的重要环节是将病毒RNA基因组，在病毒逆转录酶(Reverse transcriptase, RT)的作用下，逆转录成双链DNA。随后病毒的双链DNA经历入核和整合，整合进宿主细胞基因组中。在此过程中会产生一系列中间产物，逆转录起始产物ssDNA，ssDNA发生链跳转后继续合成的产物U3U5序列，逆转录完成产物late-RT， HIV-1病毒DNA入核后形成的2LTR环状结构（2LTR circle）以及表示病毒DNA完成整合的Alu-LTR结构。通过检测这些中间产物，不仅可以了解病毒复制的情况，还可以更深入地研究抗HIV-1药物的作用机制。 我们建立了qPCR检测上述复制中间产物的方法，采用靶点明确的药物AZT、EFV和RAL分别对ssDNA、U3U5、late-RT、2LTR以及Alu-LTR的检测方法进行验证。结果显示AZT组的ssDNA和U3U5与PC相比并无明显减少，而late-RT和2LTR则显著降低，与AZT作用于逆转录阶段、是核苷类逆转录酶抑制剂相符合。EFV组在ssDNA的合成阶段即产生了抑制作用，与EFV作用于逆转录阶段、是非核苷类逆转录酶抑制剂相符合。RAL对late-RT无抑制作用，对2LTR有富集作用，而Alu-LTR较PC明显降低，与RAL作用于整合阶段相符合。以上结果证明该方法用于药物作用靶点研究是有效可信的。 随后，我们对6个体外抗病毒活性好，但作用靶点未知的化合物进行了HIV-1复制中间产物的检测，初步研究了药物的作用机制。检测结果显示化合物1和化合物2对ssDNA的产生无明显抑制，而显著降低了late-RT的产生，判断化合物1和化合物2作用于HIV-1逆转录后期阶段。化合物3的检测结果显示，ssDNA在感染1h后即有少量减少，late-RT明显降低，表示化合物3对逆转录整个过程均有抑制作用。化合物4对 late-RT基本无抑制作用，表示化合物4作用于逆转录之后，2LTR明显降低，表示化合物4作用于入核阶段。化合物5和化合物6对late-RT和2LTR均无抑制作用，而Alu-LTR的有所降低，表示这2个化合物的作用位点在整合阶段。 综上所述，本研究成功建立了检测HIV-1复制中间产物ssDNA、U3U5、late-RT、2LTR和Alu-LTR的方法。采用建立的方法对6个抗HIV-1药物进行了初步的机制研究，研究表明检测HIV-1复制的中间产物的可以初步判断抗HIV药物的作用阶段，可以为进一步的机制研究提供方向。 |
| 英文摘要 | Due to the lack of effective vaccine to prevent HIV infection, it is urgent to find new effective and economical anti-HIV drugs to deal with HIV. The guidances for preclinical pharmacodynamics studies announced by U.S. FDA and China SFDA point out that ,it is both important to study the antiviral activity and the mechanism of anti-HIVdrugs. It is available in clinical therapy and drug resistant research. One of the most important stages of HIV-1 replication is reverse transcription. Reverse transcription converts the single-stranded viral RNA genome into a linear, double-stranded DNA copy that is ultimately integrated into host chromoaomal DNA. Several intermidiat products are produced in this precess. The first DNA product synthesized by RT is ssDNA. U3U5 DNA is synthesized after ssDNA transfer. The late-RT product is synthesized when reverse transcription is completely finished. The 2LTR circle is produced after nucleus import and the Alu-LTR structure is formed after integration. Analysing the intermediate product can not only reveal the replication process of the virus, but also help further study in the mechanism of anti-HIV drug. In this thesis, we have set up methods to detect ssDNA, U3U5, late-RT, 2LTR and Alu-LTR. The methods were evaluated by AZT, EFV and RAL. The results displayed that compared with positive control, the late-RT was inhibited by AZT while the copy of ssDNA and U3U5 didn’t decrease, which was comsistent with the mechanism of AZT. The synthesis of ssDNA was inhibited by EFV, which matched the target of EFV, the reverse transcriptase. RAL never decreased the copy number of late-RT, while making 2LTR increasing. The copy number of Alu-LTR was decreased, indicating that RAL inhibited the integration of HIV-1. These results suggested that this method was established effectively and successfully. Here, we used the methods to test six compounds whose targets were unknown. As the results of real-time PCR showed, compound1 and compound 2 had little influence on ssDNA, but sharply decreased the copy number of late-RT. The results indicated that the target of compound 1 and compound 2 was late stage of reverse transcription. As for compound 3, it reduced the quantity of ssDNA in small scale, and decrease late-RT a lot, suggesting that compound3 targeted on reverse transcription. When dealt with compound 4, late-RT had little reduction while 2LTR decreased sharply, which indicated that compound 4 inhibited the nuclear import of viral cDNA. Compound 5 and compound 6 didn’t decrease the late-RT and 2LTR, but reduced the Alu-LTR sharply, suggesting their roles in inhibiting the integration of HIV-1. In conclusion, we successfully established methods to analyse the ssDNA, U3U5, late-RT, 2LTR and Alu-LTR products in HIV-1 replication and adopted the methods to study the mechanism of anti-HIV drugs. The results showed that by analysing the intermediate products, we can assess the active stage of the ant-HIV drugs and provide imformation for further mechanism study. KEY WORDS：HIV-1; qPCR; Intermediate product; Reverse transcription; Integration; Anti-HIV drugs. |
| 语种 | 中文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10156]  |
| 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 | 陈欢. HIV-1复制中间产物检测方法的建立及在抗HIV药物研究中的初步应用[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：昆明动物研究所
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