| 题名 | 恒河猴TRIM5α 的组织分布及TRIMCyp 限制HIV-1 病毒颗粒产生的初步研究 |
| 作者 | 汤霞 |
| 学位类别 | 硕士 |
| 答辩日期 | 2009-06 |
| 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 | 恒河猴 北平顶猴 鹰猴 TRIM5α TRIMCyp HIV-1 组织分布 |
| 其他题名 | Tissue Distribution of Rhesus Monkey TRIM5α and the Research of the Restriction of TRIMCyp on the Production of HIV-1 |
| 中文摘要 | 三重基序蛋白TRIM5α(Tripartite motif protein 5 alpha)是哺乳动物细胞中一种重要的限制因子，广泛分布于各种哺乳动物细胞中。人类TRIM5α mRNA 广泛表达于人类各个组织中，并且I 型干扰素IFN-α/β/γ 均能与TRIM5α 基因启动子的ISRE 元件结合，上调TRIM5α mRNA 的表达。恒河猴(Macaca mulatta)TRIM5α 是恒河猴体内重要的限制因子。目前对恒河猴尤其是中国恒河猴TRIM5α 的组织分布以及在受到外界刺激时TRIM5α mRNA 表达量的变化研究还未见报道。本论文通过从中国恒河猴各组织中提取总RNA，以β-actin 基因作为内参照，通过逆转录PCR 检测各组织中TRIM5α mRNA 的表达。我们选择用HIV-GFP-VSVG 感染、用佛波脂(Phorbol myfismte acetate, PMA)+离子霉素(Ionomycin, Ion)，CD28 抗体+CD49d 抗体分别共刺激恒河猴PBMC，研究不同刺激对中国恒河猴TRIM5α mRNA 表达量的影响。研究发现：TRIM5α mRNA 广泛表达于恒河猴各组织中，在免疫系统和泌尿生殖系统各组织，如腹淋巴结、睾丸和附睾中表达量最高，而在神经系统各组织如大脑、脊髓中表达量比较少，在其他各组织中未见明显的表达差异。此外HIV-GFP-VSVG 感染、PMA+ Ion 与CD28 抗体+CD49d 抗体分别共刺激PBMC 均能促进PBMC TRIM5α mRNA 表达量的上调。 TRIM5α 作为恒河猴体内的最主要的限制HIV-1 感染的限制因子，除了可能通过促进HIV-1 的脱壳和阻止整合前复合物PIC(pre-integration complex)入核，恒河猴TRIM5α 还能限制HIV-1 病毒颗粒的产生。在这个过程中B30.2 结构域是非必需的，而B-box2 和Coiled-Coil 结构域起着决定性的作用。因为鹰猴(Aotes trivirgatus)TRIMCyp(omTRIMCyp) 蛋白和北平顶猴(Macaca leouina) TRIMCyp(npmTRIMCyp）蛋白的B-box2 和Coiled-Coil 结构域与恒河猴TRIM5α 的B-box2 和Coiled-Coil 具有很高的同源性，我们希望了解鹰猴TRIMCyp 蛋白和北平顶猴TRIMCyp 蛋白对HIV-1 病毒颗粒的产生是否有限制作用。本论文主要通过将质粒pNL4.3 分别与质粒pLPCX 、pLPCX-npmTRIMCyp-HA 、 pLPCX-omTRIMCyp-HA和pLPCX-rhTRIM5α-HA共转染293T细胞，通过western blot 检测细胞内Gag 蛋白和TRIM5 蛋白的表达情况，研究omTRIMCyp 蛋白和 npmTRIMCyp 蛋白对HIV-1 病毒颗粒产生的限制作用。结果表明：北平顶猴 TRIMCyp 蛋白、鹰猴TRIMCyp 蛋白都能不同程度的促进HIV-1 病毒Gag 蛋白的降解。 |
| 英文摘要 | TRIM5α is a very important restriction factor in mammal cell, distributing broadly in many kinds of mammal cells. Human TRIM5α mRNA expresses in many kinds of human tissues. Type I Interferon, IFN-α/β/γ can combine with the ISRE element of the promoter of TRIM5α gene, promoting the expression of TRIM5α mRNA. The Rhesus monkey(macaca mulatta) TRIM5α is a very important restriction factor. There is no report about the tissue distribution of TRIM5α mRNA in Rhuses monkey and the change of expression of TRIM5α mRNA when the Rhuses monkey was stimulated. We extracted the whole RNA in many kinds of tissues of Rhuses monkey, and detected the expression of TRIM5α mRNA in tissues by RT-PCR with the β-actin gene as internal reference. Furthermore, we used the HIV-GFP-VSVG to infect and the phorbol myfismte acetate(PMA)+Ionomycin(Ino), CD28 antibody+CD49d antibody to costimulate the PBMC of Rhuses monkey separately in order to study the influence of different stimuli on the expression of Rhuses monkey TRIM5α mRNA. The results showed that TRIM5α mRNA expressed broadly in many kinds of tissues of Rhuses monkey. They were expressed in highest level in tissues of immune system and urogenital system, such as lymphonodi abdominals, testicle, epididymis etc. however they were expressed fewest in nervous system, such as cerebrum, spinal cord, and there was not distinct difference in rest of the tissues. Furthermore the infection of HIV-GFP-VSVG , the costimulation of PMA+Ion, CD28 antibody+CD49d antibody to PBMC could promote the up-regulation of TRIM5α mRNA of PBMC. Rhuses monkey TRIM5α can restrict the production of HIV-1 except for restricting the reverse transcription of HIV-1 or the entry of PIC(pre-integration complex) to the cell nucleus to restrict the infection of HIV-1. The B30.2 domain is not necessary, but B-box2 and Coiled-Coil domains are indispensable during this process. Because of the conservation of the B-box2 and Coiled-Coil domains of owl monkey (Aotes trivirgatus) TRIMCyp (omTRIMCyp), pig-tailed monkey (Macaca leouina) TRIMCyp (npmTRIMCyp) and Rhuses monkey TRIM5α (rhTRIM5α) protein, we expected to know weather the omTRIMCyp protein and npmTRIMCyp protein can restrict the production of HIV-1. We cotransfected the plasmid pNL4.3 respectively with the plasmid pLPCX, pLPCX-npmTRIMCyp-HA, pLPCX-omTRIMCyp-HA and pLPCX-rhTRIM5α-HA into 293T cells. Then the expression of the intracellular Gag protein and TRIM5 protein was detected by western blot to study the restriction effect of omTRIMCyp protein and npmTRIMCyp on the production of HIV-1. The results showed that the npmTRIMCyp protein and the omTRIMCyp protein can both promote the degradation of Gag of HIV-1. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6303]  |
| 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 | 汤霞. 恒河猴TRIM5α 的组织分布及TRIMCyp 限制HIV-1 病毒颗粒产生的初步研究[D]. 北京. 中国科学院研究生院. 2009. |
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