多靶位RNA干扰对HIV-1抑制作用的研究
发布时间:2018-04-02 16:38
本文选题:人免疫缺陷病毒 切入点:多靶位RNA干扰 出处:《南开大学》2014年博士论文
【摘要】:人免疫缺陷病毒(Human Immunodeficiency Virus, HIV)是能引发人体发生获得性免疫缺陷综合症即“艾滋病”的病原体。自1981年发现以来,艾滋病及HIV感染已成为一个世界性的难题。目前还没有任何有效的疫苗来预防HIV的感染,药物治疗仍是治疗艾滋病最有效的方法。药物引发对人体的副作用和某些耐药HIV病毒株的出现已经成为艾滋病药物治疗的最大困扰。RNA干扰(RNAinterference, RNAi)已经广泛应用于抑制多种病毒(如HIV)的基因表达和感染的研究中。由于HIV-1病毒基因组的高突变性,单一的siRNA不能长期抑制HIV-1病毒的复制。为了解决这一问题,可以将RNAi的作用靶点选择在HIV-1病毒基因的高度保守区,同时进行多个RNAi靶点的联合抑制。这种联合抑制可以减少HIV-1逃脱RNA干扰的机率,达到持久的抑制效果。开展HIV-1的RNAi对艾滋病的预防和基因治疗都有着重要的实用价值。 本研究以RNAi为主线,从vpu-RNAi与宿主抗病毒因子tetherin的联合抑制,特定HIV-1人群的gag基因的单基因RNAi和多靶位RNAi三个方面来验证RNAi对HIV-1病毒的抑制效果。 Vpu是HIV-1特有的蛋白,它可以拮抗宿主抗病毒蛋白tetherin对病毒颗粒的束缚作用。为了消弱Vpu的拮抗作用并增强tetherin的抗病毒作用,我们采取了vpu-RNAi与tetherin过表达的方式联合抑制HIV-1的策略。在TZM-bl细胞中,首次采用2种慢病毒载体联合抑制HIV-1的复制。在具体实验过程中,我们以表达vpu-siRNA和tetherin的两种慢病毒以不同比例进行联合抑制HIV-1。经过对感染HIV-1的TZM-bl细胞中的Luciferase测定,vpu-siRNAi对HIV-1激活的luciferase活性抑制效率最高。实验结果证明单一siRNA可以达到比联合抑制更好的抑制效果。 HIV-1有多种病毒亚型,并且该病毒在不同地区和人群中的传播和流行情况也不一样。在确认了单一siRNA的抑制效果后,我们又以天津地区男男同性恋感染HIV-1人群的HIV-1gag基因为RNAi靶点,探讨gag-siRNAi对特定HIV-1人群的抑制水平。通过对44条来自天津MSM HIV-1感染者的gag基因序列分析,我们找出了序列保守位点,并设计了5个shRNA (gag-shRNA-1~5)。通过对gag-EGFP融合基因的抑制水平,确认了gag-shRNA-3对不同亚型的gag基因和HIV-1B/C两种亚型的感染性克隆都有较高的抑制能力。 在确认单一siRNA(vpu-siRNA和gag-siRNA)都有抑制效果的情况下,我们同时选取了HIV-1的结构基因(gag, env)、调控基因(tat)和辅助基因(vpu)作为RNAi的靶点。通过表达多个小干扰RNA (siRNA)的表达原件——dlhRNA (double long hairpin RNA)来实现HIV-1的多靶位抑制。本研究以含靶点序列的HIV-1基因和绿色荧光蛋白融合系统来检测RNAi的抑制效率。实验中分别构建了4个表达单个siRNA的短发夹RNA(shRNA),8个同时表达两个siRNA的长发夹RNA (1hRNA)和2个同时表达四个siRNA的双长发夹RNA (dlhRNA) o最后得到了抑制效果最好的双长发夹RNA表达原件dlhRNA-VGTE,并将dlhRNA表达原件导入FG12慢病毒载体。在TZM-bl细胞中,经过持续加入FG12-VGTE病毒(MOI=0.04)可以完全抑制HIV-1病毒的复制。本文中还利用FG12-VGTE慢病毒构建了表达dlhRNA-VGTE的VGTE-TZM-bl细胞系。VGTE-TZM-bl细胞可以抵抗HIV-1病毒的感染达10天之久。同时dlhRNA表达原件不会诱导细胞内干扰素通路的激活。所以,多靶位RNAi有望被用作HIV-1的基因治疗,为进一步展开HIV-1的基因治疗提供了参考依据。
[Abstract]:Human immunodeficiency virus (Human Immunodeficiency, Virus, HIV) is the human body can cause acquired immunodeficiency syndrome "AIDS" pathogens. Since its discovery in 1981, AIDS and HIV infection has become a worldwide problem. There is no effective vaccine to prevent HIV infection, drug treatment is still the most effective way to the treatment of AIDS drugs. Causing side effects on the human body and some resistant strains of HIV has become the biggest problem treatment of AIDS drugs (RNAinterference, RNAi).RNA interference has been widely used in suppressing a variety of virus (HIV) infection and gene expression studies. Due to the HIV-1 virus genome high mutation, single siRNA can not be long-term suppression of HIV-1 replication. In order to solve this problem, can be the target of RNAi and HIV-1 in high virus gene The joint inhibition of multiple RNAi targets is carried out at the same time. The joint inhibition can reduce the probability of HIV-1 escaping from RNA interference and achieve lasting inhibition effect. HIV-1 RNAi has important practical value for AIDS prevention and gene therapy.
In this study, we take RNAi as the main line to verify the inhibitory effect of RNAi on HIV-1 virus from three aspects, namely, the joint inhibition of vpu-RNAi and host antiviral factor tetherin, the single gene RNAi and multi target RNAi of gag gene in a specific HIV-1 population.
Vpu is a HIV-1 specific protein, it can antagonize the host antiviral protein tetherin binding of viral particles. In order to weaken the antagonism of Vpu and enhance the antiviral effect of tetherin, we took over expression of vpu-RNAi and tetherin combined inhibition of HIV-1 strategy. In TZM-bl cells, for the first time by using 2 kinds of lentiviral vector combined inhibition the replication of HIV-1. In the process of experiment, we pass to HIV-1 infection in TZM-bl cells by Luciferase combined inhibition of HIV-1. in different proportion to the expression of two types of virus vpu-siRNA and tetherin, vpu-siRNAi on the activation of HIV-1 luciferase inhibited the activity of the highest efficiency. Experimental results show that single siRNA can achieve better inhibitory effect than combined inhibition.
HIV-1 has a variety of virus subtypes, and the spread of the virus in different regions and populations and the epidemic situation is not the same. In recognition of the inhibitory effect of single siRNA, HIV-1gag gene and HIV-1 infection in our Tianjin area gay men for targeting RNAi, to explore the effect of gag-siRNAi on the level of specific HIV-1 populations. Through the analysis of 44 gag sequences from Tianjin MSM HIV-1 infection, we found conserved sites, and 5 shRNA (gag-shRNA-1 ~ 5). By inhibiting the level of gag-EGFP fusion gene, confirmed the inhibition ability of the infectious clone gag-shRNA-3 of different subtypes of gag gene and HIV-1B/C two subtypes type are high.
In the confirmation of single siRNA (vpu-siRNA and gag-siRNA) had inhibitory effect, we selected the structural gene of HIV-1 (GAG, Env), gene (TAT) and auxiliary gene (VPU) as the target of RNAi. The expression of a number of small interfering RNA (siRNA) on the surface of the original dlhRNA (double long hairpin RNA) multi target suppression is achieved in HIV-1. In this study, including the target sequence of HIV-1 gene and green fluorescent protein fusion system to detect the inhibition efficiency of RNAi. In the experiment we constructed 4 expression of a single siRNA short hairpin RNA (shRNA), 8 and two siRNA expression a long hairpin RNA (1hRNA) and 2 at the same time the expression of double long hairpin RNA four siRNA (dlhRNA) O finally got the best inhibitory effect of double long hairpin RNA expression in the original dlhRNA-VGTE, and the dlhRNA expression of the original into FG12 lentiviral vector. In TZM-bl cells, after adding FG12-VGTE virus (MOI=0.04) can completely inhibit the replication of the HIV-1 virus. This paper also uses the FG12-VGTE lentiviral construct VGTE-TZM-bl expression cell line.VGTE-TZM-bl dlhRNA-VGTE can resist the infection of HIV-1 virus for 10 days. At the same time, the expression of dlhRNA and activation of intracellular interferon pathway induced by the original will. Therefore, multi target RNAi is expected to be used the gene therapy of HIV-1, provide a reference basis for further gene therapy of HIV-1.
【学位授予单位】:南开大学
【学位级别】:博士
【学位授予年份】:2014
【分类号】:R512.91
【参考文献】
相关期刊论文 前4条
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4 ;The Role of HIV Replicative Fitness in Perinatal Transmission of HIV[J];Virologica Sinica;2011年03期
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