| 题名 | TRIM5α基因的分子进化及AIDS 免疫治疗相关腺病毒载体的构建 |
| 作者 | 刘红亮 |
| 学位类别 | 博士 |
| 答辩日期 | 2006-07 |
| 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 TRIM5α 分子进化 树突状细胞 HSP70 免疫治疗 |
| 其他题名 | Molecular Evolution of TRIM5α Gene and rAdenovirus Construction Related to AIDS Immunotherapy |
| 学位专业 | 动物学 |
| 中文摘要 | 本文包括两方面的研究工作。第一部分对TRIM5α基因在灵长类动物中的分子进化进行了详尽的研究，希望能从分子进化的角度认识该基因在灵长类动物抵抗病毒感染尤其是在限制HIV-1感染过程中所起的作用，并希望能够从中寻找适合用于AIDS基因治疗的基因。第二部分构建了艾滋病治疗性DC疫苗相关的腺病毒载体，包括表达HIV-1 gp120基因、表达HIV-1 gp41基因、表达HSP70基因以及仅表达绿色荧光蛋白基因的重组腺病毒，为利用HIV-1抗原刺激的DC免疫治疗AIDS的研究奠定了基础。 在长期的HIV-1和AIDS研究中，人们发现很多种旧大陆猴不能被HIV-1感染，而旧大陆猴中的平顶猴却对HIV-1有易感性，并且对SIV的感染也表现出较其它旧大陆猴更为严重的症状，TRIM5α基因的发现解释了这一“历史之谜”。人们发现旧大陆猴的TRIM5α基因具有很强的抗HIV-1的功能，是旧大陆猴抵抗HIV-1感染必须的免疫因子；并且进一步证明TRIM5α是灵长类动物中普遍存在的、具有抗多种逆转录病毒功能的天然免疫因子。我们通过对TRIM5α基因在灵长类动物中的分子进化研究发现，证明该基因在进化过程中表现出很强的达尔文正向选择作用，尤其是其负责识别逆转录病毒衣壳蛋白的SPRY结构域。TRIM5α基因表现出的正向选择很可能是灵长类动物在长期的进化过程中反复被病毒感染的选择压力造成的，这也从另一个方面证明TRIM5α基因是一个普遍存在的、具有抗病毒功能的天然免疫因子。对不同灵长类动物的TRIM5α基因序列分析发现，该基因的序列变化很大，这可能是不同灵长类动物被HIV-1感染后表现不同症状的原因；基于密码子的中性检测证明，TRIM5α基因SPRY结构域上第347、354、552位氨基酸表现出很强的正向选择，说明这些位点可能与其抗病毒功能关系紧密。另外，我们发现平顶猴的TRIM5α基因与CypA基因以一种新的方式融合，这种融合方式不同于鹰猴中该基因与CypA基因的融合，而且与具有较强抗HIV-1功能的恒河猴的TRIM5α基因比较，平顶猴的TRIM5α基因在几个与其抗病毒功能紧密相关的位点上的氨基酸也发生了变化，这可能是其被HIV-1和SIV感染后表现出较其它旧大陆猴更为严重症状的原因。 将经过HIV-1抗原刺激的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可以治疗AIDS，而且对HARRT治疗能够产生很好的协同作用；HSP70蛋白可以提高DC对抗原的加工和呈递。为了检测经HIV-1抗原刺激的DC免疫治疗AIDS的实际效果，同时检验通过HSP70增强DC对HIV-1抗原的加工和呈递功能，发现并解决这一治疗方案存在的问题，我们首先从HIV-1的基因组及带有HSP70基因的质粒上克隆了gp120、gp41和HSP70基因，测序及序列分析证明所克隆的基因没有发生影响表达的突变。然后我们分别构建了表达有关基因的重组腺病毒载体。在荧光显微镜下可以观察到感染后的细胞内绿色荧光蛋白报告基因的表达，证明成功构建了四种重组腺病毒，而PCR扩增也证明在其中三种重组腺病毒的基因组上确实分别带有gp120、gp41、HSP70基因。同时我们还构建了不表达外源基因的重组腺病毒，作为未来实验中的对照载体。未来的研究工作包括目的基因表达检测，重组腺病毒感染DC及腺病毒感染DC功能分析等。 |
| 英文摘要 | Two distinct projects involving HIV-1/AIDS were discussed in this thesis: (1) molecular evolutionary of TRIM5α, a HIV-1 infection restrictive factor in Old World monkey; (2) the construction of rAdenovirus experssing HSP 70，HIV-1 gp120, gp41, the aim is by infecting dendritic cell (DC) to stimulate DC with HIV-1 antigen and increase the processing capacity of DC, as the base of AIDS immunotherapy with DC. Most of the Old World monkeys are restrictive to HIV-1 infection, while pig-tailed monkey is permissive to both of HIV-1 and SIV infection, what is more this kind of monkey takes on more serious symptom when infected by SIV than other kinds of Old world monkey do. The cellular TRIM5α protein, a potential innate immunity factor in many kinds primate which can restrict retrovirus was recently identified as underlying HIV-1 restriction in Old World monkey cells. TRIM5α belongs to a large family of proteins containing a tripartite motif (TRIM) comprising a RING domain, one or two B-box domains, and a coiled-coil domain, TRIM5α also contains a characteristic SPRY domain at its carboxy terminus critical for determining the species-specific restriction of HIV-1 by Old World monkeys. Here by analyzing its evolutionary history, we find strong evidence for ancient positive selection in the primate TRIM5α gene, especially in the SPRY domain which could recognize the capsid of the invading retroviruses. This history suggests that TRIM5α evolution has been driven by antagonistic interactions with a variety of viruses and endogenous retroviruses that predate the origin of primate lentiviruses. Our results provide more evidence to identify TRIM5α as a widespread innate immunity factor. TRIM5α gene encoding sequences from different primate vary greatly which interpret primate represent diverse symptom after infected by HIV-1. Neutral test based on the single codon reveals amino acids in the position of 347/354/552 show strong positive selection, which could prove those amino acid positions had played an important role in the restriction of retroviruses. In the genome of owl monkey, TRIM5α gene fuse with CypA gene. In the genome of pig-tailed monkey, we found both of them had fused together in a new way, and the amino acids related close with its function had changed, which could be the main reason that pig-tailed monkey can be infected by HIV-1, and show the most serious symptom when infected by SIV. The second project focused on the construction of rAdenovirus expressing HSP 70, HIV-1 gp120, gp41, with GFP gene as indicator. After transported back to the body, DC pulsed with HIV-1 antigen can induce strong immunity against HIV-1, which can cure AIDS theoretically and cooperate with the HARRT. HSP70 protein can boost the ability of DC to process and present antigen. To verify the therapy effect of the immunity induced by DC pulsed with HIV-1 antigen to AIDS, test the ability of HSP70 protein and find out the problems in this way to treat AIDS. We initially cloned gp120 and gp41 from the genome of HIV-1IIIB, and cloned the HSP70 gene from a plasmid. The resorts of sequencing and sequence analysis prove no mutation has occurred which can affect the genes to express. then we inserted the genes to the expressing vectors, and constructed the rAdenoviruses. Expression of the report gene of GFP testified rAdenoviruses had been constructed successfully, and the resorts of PCR amplification also proved rAdenoviruses took the genes separately. At the same time we have constructed a rAdenovirus expressing no inserted gene, which will be used as a control.The further works will include the test of protein expression, rAdenoviruses infecting DC and analyzing function of DC infected by rAdenovirus etc. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6141]  |
| 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 | 刘红亮. TRIM5α基因的分子进化及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. 2006. |
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