HIV-1在不同传播方式下准种的差异性研究

发布时间：2018-01-20 14:19

本文关键词： HIV-1 垂直传播性传播准种构象敏感性凝胶电泳　出处：《第三军医大学》2005年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的准种(quasispecies)是由Eigen首先提出的用于描述同种生物遗传异质性的概念。在病毒学研究领域,准种被用于描述感染个体内同种病毒的遗传异质性(genetic heterogeneity)。准种特性已经被证实是RNA病毒具有的一种普遍的生物学特性。HIV-1是一种变异活跃的逆转录病毒,其逆转录酶在病毒基因组反向转录和正向转录过程中均缺乏自身校正功能,导致同亚型或不同亚型病毒株间基因片段重组、缺失、重复或倒置,并且随流行时间的延长,变异程度不断增大。构象敏感性凝胶电泳(conformation sensitive gel electrophoresis, CSGE)是一种很好的基因多态性分析的方法,其实质上是异源双链分析(heteroduplex analysis, HDA)的改进方法。在本研究中,将以往的CSGE的方法做了如下改进:①在聚丙烯酰胺凝胶中加入15%尿素;②甲酰胺浓度从15%提高至20%,乙二醇浓度从10%提高至15%;③通过一次预电泳优选驱动子序列。加入尿素可使条带更狭窄和更清晰,适当增加甲酰胺和乙二醇的浓度,提高了CSGE检测的敏感性,通过一次预电泳优选出的驱动子序列,可以使电泳条带间距离更大,更容易判读电泳结果。因为使用的PCR产物未作纯化,其中的非特异扩增产物对同源和异源双链的形成都有较大的影响,直接造成电泳条带模糊不清,因此我们对PCR作了严格的优化,最大限度地减少了非特异产物的干扰。性传播和垂直传播是HIV-1重要的传播方式,HIV-1通过这2种方式更换宿主。尽管从理论上讲新宿主体内的HIV-1应与传染源体内的HIV-1完全相同,但是已经有越来越多的实验证实事实并非如此。我利用CSGE的手段对不同传播方式即垂直传播和性传播感染者体内HIV-1准种进行差异性研究,了解HIV-1在传播过程中以及在新宿主体内免疫压力下准种变化,进而揭示HIV-1准种的进化规律,同时为阐明HIV-1母婴垂直传播和性传播的发生机制提供理论依据。材料和方法 1. 研究对象:母婴传播和性传播HIV-1感染者的血浆。 2. 主要试剂:QIAamp Viral RNA mini kit(Qiagen,美国);QIAGEN OneStep RT-PCR
[Abstract]:Background and purpose of the study Quasispeciesis a concept first proposed by Eigen to describe genetic heterogeneity of the same species in the field of virology. Quasispecies are used to describe genetic heterogeneity of homoviruses in infected individuals. Quasispecies characteristics have been proved to be a universal biological characteristic of RNA. HIV-1 is an active mutated retrovirus. The reverse transcriptase lacks self-correcting function in the reverse transcription and forward transcription of the virus genome, which results in the recombination, deletion, repetition or inversion of the gene fragments between the same subtype or different subtype virus strains. And with the extension of epidemic time, the degree of variation increased. Conformational sensitive gel electrophoresis conformation sensitive gel electrophoresis. CSGE is a good method for gene polymorphism analysis, which is essentially heteroduplex analysis. In this study, the previous CSGE method was modified as follows: 1 adding 15% urea to polyacrylamide gel; (2) the concentration of formamide increased from 15% to 20 and the concentration of ethylene glycol increased from 10% to 15. 3The driver sequence was selected by one pre-electrophoresis. Adding urea can make the band narrower and clearer, increase the concentration of formamide and ethylene glycol appropriately, and improve the sensitivity of CSGE detection. Through a pre-electrophoresis of the selected driver sequence, the electrophoresis bands can make the distance between the larger, easier to interpret the results of electrophoresis, because the PCR products used are not purified. The non-specific amplification products have great influence on the formation of homologous and heterogenous double strands, which directly lead to the blurring of electrophoresis bands, so we have made strict optimization of PCR. The interference of non-specific products is minimized. Sexual transmission and vertical transmission are important modes of HIV-1 transmission. HIV-1 changes the host in these two ways, although in theory the HIV-1 in the new host should be exactly the same as the HIV-1 in the source of infection. But more and more experiments have confirmed that this is not the case. I use the means of CSGE to study the difference of HIV-1 quasispecies in different transmission modes that is vertical transmission and sexually transmitted infection. To understand the changes of HIV-1 quasispecies during transmission and in vivo immune pressure of new hosts, and to reveal the evolution of HIV-1 quasispecies. At the same time, it provides theoretical basis for clarifying the mechanism of vertical transmission and sexual transmission of HIV-1. Materials and methods 1. Study subjects: plasma levels of HIV-1 infected with mother-to-child and sexually transmitted infections. 2. Main reagent: QIAamp Viral RNA mini kit, QIAGEN OneStep RT-PCR
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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