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题名	灵长类动物中TRIM5-CypA融合基因形成模式检测
作者	曹光
学位类别	硕士
答辩日期	2011-05
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	熊猴 限制因子 TRIM5α TRIM5-CypA 融合基因 杂合子基因型 HIV 限制活性
其他题名	Screening the genotypes of TRIM5-CypA fusion gene in primates
学位专业	生物化学与分子生物学
中文摘要	缺乏合适的动物模型是制约艾滋病研究取得重大突破的关键瓶颈之一。细胞内的抗病毒蛋白被称为限制因子。研究不同灵长类动物抗HIV-1宿主限制因子的存在形式及作用机制对建立合适的AIDS灵长类动物模型有十分重要的意义。TRIM5α是哺乳动物细胞中一种重要和关键的限制因子, 它以物种依赖的方式限制包括HIV-1在内的逆转录病毒的感染。TRIM5-CypA融合基因是存在于新大陆猴与旧大陆猴中的一种独特的TRIM5基因形式。 为了研究不同灵长类动物TRIM5基因的存在方式，本文对熊猴、藏酋猴、红面猴及中国恒河猴4个物种共110只灵长类动物进行了TRIM5-CypA融合模式的研究。首次发现熊猴也存在TRIM5-CypA基因融合现象。熊猴TRIMCyp融合基因形成模式类似于北平顶猴TRIMCyp融合基因模式, 即CypA假基因的cDNA序列通过逆转座方式插入到TRIM5基因的3'-UTR区域。基因序列分析表明, 该基因与北平顶猴相应基因序列高度相似；并且其TRIM5内含子6的3'-剪接位点也相应存在G-to-T突变现象(G/T)。通过RT-PCR对熊猴TRIM5-CypA融合基因的转录本进行分析，我们鉴定出熊猴TRIMCyp融合基因共有4种不同的选择性剪接产物，将它们分别命名为asmTRIMCypV1-V4。克隆丰度和序列分析结果显示，asmTRIMCypV2是优势剪接体并可能在该融合基因产物的功能中发挥作用。研究发现熊猴TRIMCyp融合基因主要转录本中外显子7和8均被剪切丢失。外显子7剪切丢失机制源于TRIM5 内含子6中3'-剪接位点的G-to-T突变。氨基酸序列比对显示，熊猴TRIMCyp融合蛋白CypA结构域中与逆转录病毒限制活性相关的氨基酸位点突变与食蟹猴TRIMCyp的CypA结构域中相应位点的突变情况相同。提示熊猴TRIMCyp可能具有与食蟹猴TRIMCyp相似的抗逆转录病毒活性。
英文摘要	The lack of appropriate animal models that utilizes HIV-1 as the challenge virus is a major impediment to HIV/AIDS research. A major reason underlying the inability of HIV-1 to replicate in nonhuman primate cells is the existence of host antiviral restriction factors. The intrinsic antiviral proteins in host cells are described as restriction factors. The understanding of restriction factors and their mechanism in different primates would undoubtedly facilitate the development of HIV/AIDS animal models. TRIM5α is an important restriction factor and can restrict the infection of several retroviruses including HIV-1 in a species-specific fashion. TRIM5-cyclophilin A (TRIMCyp) gene is an unusual TRIM5 locus found in New World and Old World monkeys. The different TRIMCyp genotypes of four primates (110 samples) including assam macaque (Macaca assamensis), tibetan macaque (Macaca thibetana), stump-tailed macaque (Macaca arctoides) and Chinese rhesus macaques (Macaca mulatta) were studied in this paper. We firstly found that TRIM5-CypA fusion gene exist in Macaca assamensis. The TRIMCyp of Macaca assamensis also results from the retrotransposition of CypA pseudogene cDNA into 3'-UTR of TRIM5 gene like TRIMCyp of Macaca leonina. Moreover, there is an extremely high sequence homology between TRIMCyp genes from Macaca assamensis and Macaca leonina. Besides, we also found the G-to-T mutation (G/T) in the 3'-splicing site of TRIM5 intron 6, which was identical to Macaca leonina. Furthermore, We identified four different alternative splicing isoforms of TRIMCyp gene from Macaca assamensis through RT-PCR and sequencing assays and named them as asmTRIMCypV1-V4 respectively. The asmTRIMCypV2 as the major isoform may involved in the function of the fusion gene product. More work indicated that both exons 7 and 8 were spliced out in the major splicing isoforms during the transciption of the fusion gene. The G-to-T mutation in the 3’-splicing site of TRIM5 intron 6 should prevent the inclusion of exon 7 during splicing. Finally, we performed the alignment of putative CypA animo acid sequences of species variants of primates TRIMCyps and found that both TRIMCyps form Macaca assamensis and Macaca fascicularis with the same amino acid mutation sites related to the restriction of different retroviruses in their CypA domain, which suggested that TRIMCyps form Macaca assamensis and Macaca fascicularis may have the similar restriction activity for protecting the host from various retroviruses infection.
语种	中文
公开日期	2011-08-26
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/353002/6725]
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	曹光. 灵长类动物中TRIM5-CypA融合基因形成模式检测[D]. 北京. 中国科学院研究生院. 2011.

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