AIDS 免疫治疗相关腺病毒载体的构建
文献类型：学位论文
| 作者 | 王路 |
| 学位类别 | 硕士 |
| 答辩日期 | 2008-03 |
| 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 AIDS gp140 tat 免疫治疗 DC |
| 其他题名 | Construction of Recombinant Adenovirus Related to AIDS Immunotherapy |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 艾滋病（AIDS）是人类免疫缺陷病毒（HIV）感染后引起的一种严重危害人类健康的致死性传染病。抗HIV药物挽救和延长了很多HIV感染者的生命，提高了其生活质量，但是仍然不能治愈AIDS和预防传播。最终有效控制HIV传播和感染的方法可能仍将依赖于HIV疫苗的应用。HIV感染对感染者以及社会造成的灾难性后果使得发展一个有效的艾滋病疫苗变得尤为紧迫和重要。 负载HIV-1抗原的DC回输到HIV-1感染者体内可以诱导产生较强的抗HIV-1细胞免疫反应，这种免疫反应理论上和临床试验都表明治疗AIDS有效，而且对HARRT治疗能够产生很好的协同作用。我们拟用感染了重组腺病毒的DC，回输到HIV-1感染者体内，期望可以较好地控制病毒复制和阻止感染。为此，本研究我们制备了重组腺病毒vAd-gp140、vAd-tat和vAd-gp140-tat，为后续研究奠定基础。 结构蛋白Env是激发抗体反应的抗原，由于Env全长有较大细胞毒性，本文采用了截短的gp140分子，删除了gp41的胞内段，降低了gp140蛋白的细胞毒性。同时保留了gp41的跨膜区，表达的蛋白可被正确地锚定在细胞表面，提高蛋白的免疫原性。将gp140分子克隆到复制缺陷型的腺病毒载体中，用Wester Blotting方法检测到gp140在293细胞中的表达。 有效的抗HIV-1的疫苗应该能够同时激发针对多种亚型病毒株的细胞和体液免疫反应。早期病毒蛋白激发的CTL应答在控制病毒载量上更为有效，而且Tat蛋白的重要免疫抗原表位和功能区域在不同HIV-1病毒株之间是高度保守的。Tat蛋白的多种生物学功能使得它成为较强的免疫原、共抗原和佐剂，激发T细胞抗原表位的Th1型反应和CTL反应，扩大体内T细胞识别的抗原表位谱，提高T细胞特异性免疫反应。本文扩增了HIV-1ⅢB的tat基因，克隆到复制缺陷性的腺病毒中，构建了重组腺病毒vAd-tat，并在293细胞中表达了分子量大小为15kDa的蛋白。 早期蛋白和结构蛋白的联合免疫能够全面地控制病毒复制，在动物实验中一定程度上保护了猴子。我们将已得到表达的gp140和tat基因进行融合表达。利用融合PCR技术，扩增gp140和tat的融合基因，构建携有HIV-1 gp140-tat融合基因的重组腺病毒vAd-gp140-tat。gp140-tat在293细胞中的融合表达还需要进一步验证。 下一步的工作是将构建好的重组腺病毒感染DC，检测外源基因在DC中的表达水平，对DC表面分子表型的影响以及对DC功能的影响. |
| 英文摘要 | Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. The urgency of designing an effective, safe, inexpensive and easily administrable vaccine to protect people from HIV and/or AIDS is absolute priority and it has turned out to be enormous challenge for the scientific community. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses that will result in enduring, broadly protective immunity, yet an efficacious vaccine remains elusive. DCs pulsed with antigen as a therapeutic DC vaccine for AIDS and delivered into body can induce strong cell-mediated immune response against HIV-1, which can control virus replication and delay disease progress to AIDS. We presumed DCs infected with recombinant Adenovirus expressing HIV-1 gene and delivered into body would control viral replication and prevent infection. In this thesis, we focused on the construction of recombinant Adenovirus expressing HIV-1 gp140, tat and gp140-tat, with GFP gene as a indicator which was included in the shuttle vector pTrack-CMV and transcriped under the control of the CMV promotor. Structural protein Env could induce neutralizing antibodies to prevent infection. Truncated gp140 was selected in this study, which have a deletion in COOH-terminal cytoplasmic for the toxicity of full Env (gp160) and transmembrane domain was retained to mimic native surface glycoprotein to elicite significantly broader immunity than soluble forms of envelope. Meanwhile, Tat-specific immune responses elicited by prophylactic vaccine can potentially have a critical impact on HIV transmission and replication. Humoral and cellular responses directed against Tat correlate with the non-progression to AIDS. We cloned gp140 and tat from plasmid pMET-HXB2-env and the genome of HIV-1IIIB respectively, and then recombine them into a DNA fragment by overlap PCR, which were inserted into the expressing vectors. The resulting recombinant plasmids, designated prAd-tat, prAd-gp140, prAd-gp140-tat, were digested by Pac I and then transfected into packaging cell lines 293 cells. The infectivity of recombinant Adenovirus was dictated by expression of the report gene of GFP. HIV-1 gp140, tat and gp140-tat gene could be detected in the supernatants of infected 293 cells by PCR. Their expression were detected in 293 cells by Western Blotting. Finally, HIV-1 genes expression was identified in Western Blooting except for recombinant gene gp140-tat. At the same time, we constructed an Adenovirus without inserted gene, which could be used as a control. The further works will include phenotype changes on the DC and analyzing function of DC infected with various recombinant Adenovirus, in addtion to their expression in the DC. |
| 语种 | 中文 |
| 公开日期 | 2010-10-13 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6079]  |
| 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 | 王路. AIDS 免疫治疗相关腺病毒载体的构建[D]. 北京. 中国科学院研究生院. 2008. |
入库方式： OAI收割
来源：昆明动物研究所
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