中国饲养食蟹猴TRIM5基因多态性研究

发布时间：2018-04-27 18:48

本文选题：食蟹猴 + AIDS　；参考：《华南理工大学》2014年硕士论文

【摘要】：艾滋病已经成为我们人类在21世纪面临的最严峻难题之一，是严重威胁人类健康的重大疾病，已在全世界造成巨大经济损失和产生严重社会问题。为更好地揭示艾滋病的发病机制和研发治疗药物及疫苗，非人灵长类AIDS疾病模型是必不可少的工具。研究显示食蟹猴基因组与人类基因组同源性极高，且食蟹猴模型能更好地模拟人类疾病历程；同时食蟹猴资源极为丰富，我国即有大量的食蟹猴养殖场，因此，更有助于食蟹猴AIDS模型建立，食蟹猴AIDS模型能更好地对AIDS进行研究。研究发现，在旧大陆猴中TRIM5α起抗逆转录病毒作用，能够限制HIV-1的活性。TRIMCyp融合基因是另一个抗HIV-1因子研究热点。不同物种中CypA插入到TRIM5基因中的位置不同，而在旧大陆猴TRIMCyp融合基因是CypA假基因cDNA序列在LINE-1介导下以逆转录转座的方式插入至TRIM5基因的3'非翻译区形成。大量研究发现TRIMCyp融合基因在不同灵长类动物中具有地域、基因频率、基因型以及抗逆转病毒效应的差异。研究也发现食蟹猴TRIMCyp蛋白主要单体型(DK)具有限制HIV-1功能，但是不能限制HIV-2；而TRIMCyp次要单体型（NE）能够限制HIV-2，却不能限制HIV-1感染，因此携带TRIMCypNE单体型的食蟹猴有可能构建成为HIV-1感染的动物模型。食蟹猴TRIMCyp基因存在状况及其多态性在东南亚几个国家或地区虽已经被初步调查，，而在中国也有部分研究，但是中国境内饲养场食蟹猴的TRIMCyp基因整体存在情况还没有明确阐明。本研究基于中国丰富的食蟹猴资源，对中国5个省份11个养殖场的食蟹猴(Macaca fascicularis)繁殖种群进行随机采样，共采集1594个食蟹猴血液样品，对其TRIMCyp基因进行了研究，研究结果如下：（1）通过文献阅读，从已报道的TRIMCyp融合情况分析CypA插入位置，设计出食蟹猴TRIMCyp筛选引物，以PCR方法对中国饲养食蟹猴TRIMCyp融合基因进行筛查。研究结果显示，我国境内各饲养场食蟹猴的TRIMCyp融合基因，主要是以TRIM5/TRIMCyp杂合子的形式出现(87.60%)，且各猴场食蟹猴TRIMCyp融合基因存在状况基本一致，仅略有差异(7.65%～19.79%)。但在东南亚不同国家或地区的调查结果TRIM5/TRIMCyp杂合子的频率是53.16%，且TRIMCyp的基因频率甚至高达100%(Philippines)，远远高于中国境内的平均水平的13.36%，可能原因是其从1978年就开始建立遗传封闭群。此外，本文对带有TRIMCyp融合基因的食蟹猴个体CypA测序结果进行了校准、比对和分析，统计了中国饲养食蟹猴TRIMCyp蛋白主要单体型(DK)和次要单体型(NE)存在情况。研究结果发现主要单体型TRIMCyp(DK)占95.07%；而次要单体型TRIMCyp(NE)的食蟹猴个体很少，NE单倍型频率仅为4.93%，且只有一个为TRIMCyp(NE)纯合子，显著低于东南亚三个国家食蟹猴的NE单倍型频率(11.1%～14.3%)。次要单体型(NE)能被HIV-1感染，而人类AIDS疾病多由逆转录病毒HIV-1引起，因此携带TRIMCyp NE单体型的食蟹猴有可能构建成为HIV-1感染的动物模型。通过在食蟹猴饲养基地建立封闭群，有可能构建更多TRIMCyp(NE)纯合子食蟹猴个体，从而更好地促进HIV-1发病机制研究。该研究为进一步开展食蟹猴HIV-1动物模型和发病机制提供了基础信息。（2）本研究先在TRIMCyp食蟹猴小群体中进行多态性初筛，通过比较分析发现TRIM5基因仅外显子7和8存在与CypA插入完全连锁的单核苷酸多态性，并在食蟹猴、恒河猴、平顶猴和藏酋猴中对TRIM5基因外显子7和8进行了大量验证研究，结果发现了8个新的与CypA插入相关的SNP位点，且这些位点多分布于外显子8的5'端，其中两个SNP位点倾向于形成一个外显子拼接沉默子(ESS)序列，这可能有助于解释外显子8在所有TRIMCyp拼接体中均不存在。TRIMCyp选择性拼接相关拼接位点多态性分析结果显示，除内含子6的3'拼接位点存在突变外，其余拼接位点均不存在突变，揭示多种拼接体的存在可能是误切的结果。
[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.

【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91

【参考文献】