本文選題：食蟹猴 + AIDS�。� 參考：《華南理工大學(xué)》2014年碩士論文

【摘要】：艾滋病已經(jīng)成為我們?nèi)祟愒?1世紀面臨的最嚴峻難題之一，是嚴重威脅人類健康的重大疾病，已在全世界造成巨大經(jīng)濟損失和產(chǎn)生嚴重社會問題。為更好地揭示艾滋病的發(fā)病機制和研發(fā)治療藥物及疫苗，非人靈長類AIDS疾病模型是必不可少的工具。研究顯示食蟹猴基因組與人類基因組同源性極高，且食蟹猴模型能更好地模擬人類疾病歷程；同時食蟹猴資源極為豐富，我國即有大量的食蟹猴養(yǎng)殖場，因此，更有助于食蟹猴AIDS模型建立，食蟹猴AIDS模型能更好地對AIDS進行研究。研究發(fā)現(xiàn)，在舊大陸猴中TRIM5α起抗逆轉(zhuǎn)錄病毒作用，能夠限制HIV-1的活性。TRIMCyp融合基因是另一個抗HIV-1因子研究熱點。不同物種中CypA插入到TRIM5基因中的位置不同，而在舊大陸猴TRIMCyp融合基因是CypA假基因cDNA序列在LINE-1介導(dǎo)下以逆轉(zhuǎn)錄轉(zhuǎn)座的方式插入至TRIM5基因的3'非翻譯區(qū)形成。大量研究發(fā)現(xiàn)TRIMCyp融合基因在不同靈長類動物中具有地域、基因頻率、基因型以及抗逆轉(zhuǎn)病毒效應(yīng)的差異。研究也發(fā)現(xiàn)食蟹猴TRIMCyp蛋白主要單體型(DK)具有限制HIV-1功能，但是不能限制HIV-2；而TRIMCyp次要單體型（NE）能夠限制HIV-2，卻不能限制HIV-1感染，因此攜帶TRIMCypNE單體型的食蟹猴有可能構(gòu)建成為HIV-1感染的動物模型。 食蟹猴TRIMCyp基因存在狀況及其多態(tài)性在東南亞幾個國家或地區(qū)雖已經(jīng)被初步調(diào)查，，而在中國也有部分研究，但是中國境內(nèi)飼養(yǎng)場食蟹猴的TRIMCyp基因整體存在情況還沒有明確闡明。本研究基于中國豐富的食蟹猴資源，對中國5個省份11個養(yǎng)殖場的食蟹猴(Macaca fascicularis)繁殖種群進行隨機采樣，共采集1594個食蟹猴血液樣品，對其TRIMCyp基因進行了研究，研究結(jié)果如下： （1）通過文獻閱讀，從已報道的TRIMCyp融合情況分析CypA插入位置，設(shè)計出食蟹猴TRIMCyp篩選引物，以PCR方法對中國飼養(yǎng)食蟹猴TRIMCyp融合基因進行篩查。研究結(jié)果顯示，我國境內(nèi)各飼養(yǎng)場食蟹猴的TRIMCyp融合基因，主要是以TRIM5/TRIMCyp雜合子的形式出現(xiàn)(87.60%)，且各猴場食蟹猴TRIMCyp融合基因存在狀況基本一致，僅略有差異(7.65%～19.79%)。但在東南亞不同國家或地區(qū)的調(diào)查結(jié)果TRIM5/TRIMCyp雜合子的頻率是53.16%，且TRIMCyp的基因頻率甚至高達100%(Philippines)，遠遠高于中國境內(nèi)的平均水平的13.36%，可能原因是其從1978年就開始建立遺傳封閉群。此外，本文對帶有TRIMCyp融合基因的食蟹猴個體CypA測序結(jié)果進行了校準、比對和分析，統(tǒng)計了中國飼養(yǎng)食蟹猴TRIMCyp蛋白主要單體型(DK)和次要單體型(NE)存在情況。研究結(jié)果發(fā)現(xiàn)主要單體型TRIMCyp(DK)占95.07%；而次要單體型TRIMCyp(NE)的食蟹猴個體很少，NE單倍型頻率僅為4.93%，且只有一個為TRIMCyp(NE)純合子，顯著低于東南亞三個國家食蟹猴的NE單倍型頻率(11.1%～14.3%)。次要單體型(NE)能被HIV-1感染，而人類AIDS疾病多由逆轉(zhuǎn)錄病毒HIV-1引起，因此攜帶TRIMCyp NE單體型的食蟹猴有可能構(gòu)建成為HIV-1感染的動物模型。通過在食蟹猴飼養(yǎng)基地建立封閉群，有可能構(gòu)建更多TRIMCyp(NE)純合子食蟹猴個體，從而更好地促進HIV-1發(fā)病機制研究。該研究為進一步開展食蟹猴HIV-1動物模型和發(fā)病機制提供了基礎(chǔ)信息。 （2）本研究先在TRIMCyp食蟹猴小群體中進行多態(tài)性初篩，通過比較分析發(fā)現(xiàn)TRIM5基因僅外顯子7和8存在與CypA插入完全連鎖的單核苷酸多態(tài)性，并在食蟹猴、恒河猴、平頂猴和藏酋猴中對TRIM5基因外顯子7和8進行了大量驗證研究，結(jié)果發(fā)現(xiàn)了8個新的與CypA插入相關(guān)的SNP位點，且這些位點多分布于外顯子8的5'端，其中兩個SNP位點傾向于形成一個外顯子拼接沉默子(ESS)序列，這可能有助于解釋外顯子8在所有TRIMCyp拼接體中均不存在。TRIMCyp選擇性拼接相關(guān)拼接位點多態(tài)性分析結(jié)果顯示，除內(nèi)含子6的3'拼接位點存在突變外，其余拼接位點均不存在突變，揭示多種拼接體的存在可能是誤切的結(jié)果。
[Abstract]:AIDS has become one of the most serious problems facing us in the twenty-first Century. It is a major disease that seriously threatens human health. It has caused huge economic losses and serious social problems all over the world. To better reveal the pathogenesis of AIDS and the development and treatment of drugs and vaccines, the non human primate AIDS disease model is not necessary. The research shows that the cynomolgus monkey genome is very homologous to the human genome, and the cynomolgus monkey model can better simulate the human disease process. At the same time, cynomolgus monkey resources are very rich, and there are a lot of crab monkey breeding farms in China. Therefore, it is more conducive to the establishment of the AIDS model of cynomolgus monkey and the AIDS model of cynomolgus monkey is better for AIDS. The study found that TRIM5 alpha plays an antiretroviral action in old continental monkeys and can restrict the HIV-1 active.TRIMCyp fusion gene to be another anti HIV-1 factor research hotspot. The location of CypA in the TRIM5 gene in different species is different, while the TRIMCyp fusion gene of the old continental monkey is the cDNA sequence of the CypA pseudogenes in LINE-1 A reverse transcriptase transposing is inserted into the 3'non translation region of the TRIM5 gene. A large number of studies have found that the TRIMCyp fusion gene has region, gene frequency, genotype and antiretroviral effect in different primates. The study also found that the TRIMCyp protein master haplotype (DK) of cynomolgus macaque has the function of limiting the HIV-1 function, but it does not. HIV-2 can be restricted, and TRIMCyp minor haplotype (NE) can limit HIV-2, but it does not restrict HIV-1 infection, so carrying TRIMCypNE haplotype cynomolgus is likely to construct an animal model of HIV-1 infection.
The existence and polymorphism of TRIMCyp gene in cynomolgus macaque have been preliminarily investigated in several countries or regions in Southeast Asia, but there are also some studies in China. However, the overall status of TRIMCyp gene in cynomolgus monkeys in China is not clearly stated. This study is based on China's rich cynomolgus monkey resources and 11 of China's 5 provinces. The breeding population of Macaca fascicularis was sampled randomly. The blood samples of 1594 cynomolgus monkeys were collected, and the TRIMCyp gene was studied. The results were as follows:
(1) through the literature reading and the analysis of the CypA insertion position of the reported TRIMCyp fusion, the TRIMCyp screening primers for cynomolgus monkeys were designed. The PCR method was used to screen the TRIMCyp fusion gene of cynomolgus macaque in China. The results showed that the TRIMCyp fusion gene of cynomolgus monkeys in the domestic farms was mainly the TRIM5/TRIMCyp heterozygote. The form appeared (87.60%), and the existence of TRIMCyp fusion gene in macaque monkeys was basically the same, only slightly different (7.65% to 19.79%). However, the frequency of TRIM5/TRIMCyp heterozygotes was 53.16% in different countries or regions in Southeast Asia, and the gene frequency of TRIMCyp was even as high as 100% (Philippines), far higher than the average in China. The possible reason for the 13.36% level is that the genetic closure group has been established since 1978. In addition, this paper calibrated the CypA sequencing results of the cynomolgus monkey with TRIMCyp fusion gene, compared and analyzed the existence of the major haplotype (DK) and secondary haplotype (NE) of the Chinese crab macaque TRIMCyp protein. The results were found to be found. The main haplotype TRIMCyp (DK) accounted for 95.07%, while the secondary monosomatotype TRIMCyp (NE) had few individuals, NE haplotype frequency was only 4.93%, and only one was TRIMCyp (NE) homozygote, significantly lower than the NE haplotype frequency (11.1% ~ 14.3%) of the three countries in Southeast Asia (11.1% to 14.3%). Minor haplotype (NE) could be infected by HIV-1, while AIDS diseases in human beings were much more. It is caused by retrovirus HIV-1, so TRIMCyp NE haplotype cynomolgus can construct an animal model of HIV-1 infection. It is possible to build more TRIMCyp (NE) homozygous cynomolgus monkeys by setting up a closed colony at the feeding base of cynomolgus macaque, so as to better promote the study of the pathogenesis of HIV-1. This study is to further develop food. HIV-1 provides basic information for animal models and pathogenesis of cynomolgus monkeys.
(2) this study first screened the polymorphism of the small group of TRIMCyp cynomolgus monkeys. Through comparative analysis, we found that only exons 7 and 8 of the TRIM5 gene had a single nucleotide polymorphism with CypA insertion, and a large number of tests were carried out on the exon 7 and 8 of the TRIM5 gene in cynomolgus monkey, Ganges RIver monkey, flat top monkey and Tibetan macaque. The results found that 8 A new SNP locus associated with CypA insertion, and these loci are mostly distributed at the 5'end of exon 8, and two of the SNP loci tend to form an exon splicing silencer (ESS) sequence, which may help explain that exon 8 does not exist in all TRIMCyp splicing related splice polymorphism analysis junctions of the.TRIMCyp selective splicing. The results showed that there were no mutations in other splice sites except for the 3'splicing site of intron 6, revealing that the existence of multiple splicing bodies might be the result of miscut.

【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.91

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