北平顶猴血清免疫学指标检测及其APOBEC3家族基因克隆和抗HIV-1研究

发布时间：2018-01-12 16:44

本文关键词：北平顶猴血清免疫学指标检测及其APOBEC3家族基因克隆和抗HIV-1研究　出处：《昆明理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着生物医学研究的深入,灵长类动物模型在医学研究领域的重要性愈发明显。在研究艾滋病、麻疹、疟疾、肝炎和神经退行性疾病等重大人类疾病以及认知等基础研究方面,灵长类动物是首选或者唯一的实验动物,其研究成果更容易转化为临床应用,可以大大降低新药研发的风险。与其他非人灵长类动物相比,猕猴属的平顶猴(pig-tailed macaques, Macaca nemestrina group, PTM)、恒河猴(rhesus macaques, Macaca mulatta, RM)与食蟹猴(cynomolgus macaques, Macaca fascicularis, CM)在人类生物医学研究领域应用最为广泛,其中平顶猴是唯一对HIV-1易感的旧大陆猴,被广泛用于HIV-1的致病机制、药物评价及疫苗等AIDS研究领域,此外平顶猴在其它感染性疾病、神经生理及行为认知等研究领域也得到了广泛应用。目前,平顶猴被分为3个种：南平顶猴(Southern pig-tailed macaque, M. nemestrina)、北平顶猴(Northern pig-tailed macaque, M. leonina, NPM)和明打威猴(Mentawai macaque, M. pagensis)。了解北平顶猴的基础生物学数据,不仅对于北平顶猴健康状态的判定、疾病的发生与发展、疾病的治疗具有重要的参考价值,而且对于北平顶猴的饲养管理和疾病模型研究也具有重要的指导意义。尽管平顶猴能够感染HIV-1,但是其在平顶猴体内的复制水平受到明显抑制,这极大地限制了HIV-1感染平顶猴模型在艾滋病疫苗、药物筛选及致病机制等研究领域的应用。感染HIV-1的平顶猴体内的HIV-1病毒存在大量的G到A突变,其突变频率远远超过了自发突变的频率,提示APOBEC3家族成员在限制HIV-1在平顶猴体内的复制方面也许发挥了重要的作用。我们之前报道过有关北平顶猴血液学和血液生化指标参考值,但是关于北平猴血清免疫球蛋白,补体与C反应蛋白(CRP)等免疫学常规指标的基础数据还未见国内外报道。本研究对不同性别与年龄段健康实验北平顶猴的血清IgA、IgG、IgM、 C3、C4及CRP进行了测定与分析,结果表明人工饲养的北平顶猴血清IgA、IgM、 C3、C4水平不随年龄发生变化,而血清IgG水平随年龄增长有增加的趋势(r=0.202；P=0.045)。血清IgG、IgA、IgM、C3水平在雌性中明显高于雄性(P0.05),而血清C4水平在雌雄间差异不显著(P=0.777)。此外,我们发现血清C3与C4水平间有很强的正相关性(r=0.700；P0.0001)。该研究为北平顶猴的饲养与生物医学研究提供了一些较重要的血清免疫球蛋白、补体的基础数据,北平顶猴免疫学相关数据的进一步检测和完善将会促进北平顶猴在感染性疾病、免疫学等生物医学研究领域的有效应用。此外,本研究成功克隆了北平顶猴APOBEC3A-APOBEC3H基因,序列比对及核酸一致性分析发现北平顶猴的APOBEC3A-APOBEC3H基因蛋白编码区序列与恒河猴、食蟹猴、南平顶猴及人的相应基因的序列的一致性高达890%～99%,尤其是APOBEC3G与APOBEC3F基因其蛋白编码区的核酸序列与恒河猴及南平顶猴相应基因序列的一致性高达99%。另外,本研究对北平顶猴APOBEC3A-APOBEC3H基因的体外抗HIV-1活性进行了研究,结果显示北平顶猴APOBEC3G/F的抗HIV-1的活性最强,APOBEC3B/D抗HIV-1的活性次之,APOBEC3H的抗HIV-1活性比APOBEC3B/D弱,APOBEC3A/C没有表现出抗HIV-1的活性。本研究提示APOBEC3家族中APOBEC3G与APOBEC3F也许是限制HIV-1在平顶猴体内复制的重要宿主限制因子之一,对HIV-1感染的平顶猴模型的优化提供了有参考价值的信息。
[Abstract]:Along with the development of biomedical research, the primate animal model of importance in the field of medical research is becoming increasingly apparent. In the study of AIDS, measles, malaria, hepatitis and basic research of neurodegenerative diseases and other major human diseases and cognition, primate animal or animal is preferred only, the results are more easily transformed into clinical application, can greatly reduce the risk of drug development. Compared with other non-human primate animal, Macaca monkfy (pig-tailed macaques, the Macaca nemestrina group, PTM), Monkey (rhesus macaques, Macaca Ganges RIver mulatta, RM) and cynomolgus monkeys (cynomolgus macaques, Macaca fascicularis, CM) in the field of Applied Research in the biomedical human widely, which is the only monkfy susceptible to HIV-1 the Old World monkey, is widely used in the pathogenic mechanism of HIV-1 and AIDS on drug evaluation and vaccine In addition, monkfy in other infectious diseases, neurophysiological and cognitive behavioral research has been widely used. At present, monkfy are divided into 3 types: Nanping top Monkey (Southern pig-tailed macaque, M. nemestrina) (Northern pig-tailed, Beijing top monkey macaque, M. leonina, NPM) and the Mentawai macaque (Mentawai macaque, M. pagensis). To understand the basic biological data of Peking monkey, not only to determine the health status of the Peking monkey, the occurrence and development of disease, which has important reference value in the treatment of disease, but also has an important guiding significance for the study of feeding management and disease model of Peking monkey. Although monkfy to HIV-1 infection, but the replication level monkfy in vivo was inhibited, which greatly limits the HIV-1 infection monkfy model in AIDS vaccine, drug screening and pathogenic mechanism Study on the application of HIV-1. Flat monkeys infected with the HIV-1 virus, there are a large number of G to A mutation, the mutation frequency is far more than the frequency of spontaneous mutations, suggesting that APOBEC3 family members in the limit of HIV-1 in monkfy replication in vivo may play an important role. We have previously reported the Peking monkey blood and the blood biochemical indexes of the reference value, but about Beijing monkey serum immunoglobulin, complement C and C-reactive protein (CRP) based data there is no conventional indicators of immunology reported at home and abroad. The levels of IgA, the research on the Beijing top monkey with different gender and age healthy experiment of IgG, IgM, C3, C4 and CRP were measured and analyzed. The results show that the artificial feeding Peking monkey serum IgA, IgM, C3, C4 levels do not change with age, and the serum IgG levels with age increasing (r=0.202; P =0.045). Serum IgG, IgA, IgM, C3 levels in females was significantly higher than that of male (P0.05), and the serum C4 levels in female and male with no significant difference (P=0.777). In addition, we found that serum C3 and C4 level, there is a strong positive correlation between (r=0.700; P0.0001). This study provided some serum immunoglobulin is more important as Peking monkey breeding and bio medical research, basic data complement, Beijing top further detection of relevant data and improve the monkey will promote the immunological Peking monkey in infectious diseases, the effective application of immunology and other biomedical research field. In addition, this study successfully cloned Peking monkey APOBEC3A-APOBEC3H gene, nucleic acid sequence alignment and consensus analysis found that the encoding sequence of APOBEC3A-APOBEC3H protein and the Ganges RIver Peking monkey monkey, Macaca fascicularis, consensus sequences corresponding genes in Nanping and the top of the monkey is up to 890% to 99%, especially APOBE The consistency of C3G nucleotide sequence of APOBEC3F gene and its protein encoding region and Ganges RIver and Nanping top the corresponding gene sequence of monkey monkey up to 99%. in the study of Peking monkey APOBEC3A-APOBEC3H gene in vitro anti HIV-1 activity were studied, the results showed that the anti HIV-1 activity of the strongest Peking monkey APOBEC3G/F, APOBEC3B/D anti HIV-1 activity second, APOBEC3H anti HIV-1 activity was weaker than APOBEC3B/D, APOBEC3A/C showed no anti HIV-1 activity. This study suggests that the APOBEC3 family APOBEC3G and APOBEC3F may be one of the main limiting factor in limiting HIV-1 important places monkfy replication, provides valuable information to optimize monkfy model of HIV-1 infection.

【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6;R512.91

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