本文選題：人免疫缺陷病毒　切入點(diǎn)：多靶位RNA干擾　出處：《南開大學(xué)》2014年博士論文

【摘要】：人免疫缺陷病毒(Human Immunodeficiency Virus, HIV)是能引發(fā)人體發(fā)生獲得性免疫缺陷綜合癥即“艾滋病”的病原體。自1981年發(fā)現(xiàn)以來,艾滋病及HIV感染已成為一個世界性的難題。目前還沒有任何有效的疫苗來預(yù)防HIV的感染,藥物治療仍是治療艾滋病最有效的方法。藥物引發(fā)對人體的副作用和某些耐藥HIV病毒株的出現(xiàn)已經(jīng)成為艾滋病藥物治療的最大困擾。RNA干擾(RNAinterference, RNAi)已經(jīng)廣泛應(yīng)用于抑制多種病毒(如HIV)的基因表達(dá)和感染的研究中。由于HIV-1病毒基因組的高突變性,單一的siRNA不能長期抑制HIV-1病毒的復(fù)制。為了解決這一問題,可以將RNAi的作用靶點(diǎn)選擇在HIV-1病毒基因的高度保守區(qū),同時進(jìn)行多個RNAi靶點(diǎn)的聯(lián)合抑制。這種聯(lián)合抑制可以減少HIV-1逃脫RNA干擾的機(jī)率,達(dá)到持久的抑制效果。開展HIV-1的RNAi對艾滋病的預(yù)防和基因治療都有著重要的實(shí)用價值。 本研究以RNAi為主線,從vpu-RNAi與宿主抗病毒因子tetherin的聯(lián)合抑制,特定HIV-1人群的gag基因的單基因RNAi和多靶位RNAi三個方面來驗(yàn)證RNAi對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對病毒顆粒的束縛作用。為了消弱Vpu的拮抗作用并增強(qiáng)tetherin的抗病毒作用,我們采取了vpu-RNAi與tetherin過表達(dá)的方式聯(lián)合抑制HIV-1的策略。在TZM-bl細(xì)胞中,首次采用2種慢病毒載體聯(lián)合抑制HIV-1的復(fù)制。在具體實(shí)驗(yàn)過程中,我們以表達(dá)vpu-siRNA和tetherin的兩種慢病毒以不同比例進(jìn)行聯(lián)合抑制HIV-1。經(jīng)過對感染HIV-1的TZM-bl細(xì)胞中的Luciferase測定,vpu-siRNAi對HIV-1激活的luciferase活性抑制效率最高。實(shí)驗(yàn)結(jié)果證明單一siRNA可以達(dá)到比聯(lián)合抑制更好的抑制效果。 HIV-1有多種病毒亞型,并且該病毒在不同地區(qū)和人群中的傳播和流行情況也不一樣。在確認(rèn)了單一siRNA的抑制效果后,我們又以天津地區(qū)男男同性戀感染HIV-1人群的HIV-1gag基因?yàn)镽NAi靶點(diǎn),探討gag-siRNAi對特定HIV-1人群的抑制水平。通過對44條來自天津MSM HIV-1感染者的gag基因序列分析,我們找出了序列保守位點(diǎn),并設(shè)計了5個shRNA (gag-shRNA-1～5)。通過對gag-EGFP融合基因的抑制水平,確認(rèn)了gag-shRNA-3對不同亞型的gag基因和HIV-1B/C兩種亞型的感染性克隆都有較高的抑制能力。 在確認(rèn)單一siRNA(vpu-siRNA和gag-siRNA)都有抑制效果的情況下,我們同時選取了HIV-1的結(jié)構(gòu)基因(gag, env)、調(diào)控基因(tat)和輔助基因(vpu)作為RNAi的靶點(diǎn)。通過表達(dá)多個小干擾RNA (siRNA)的表達(dá)原件——dlhRNA (double long hairpin RNA)來實(shí)現(xiàn)HIV-1的多靶位抑制。本研究以含靶點(diǎn)序列的HIV-1基因和綠色熒光蛋白融合系統(tǒng)來檢測RNAi的抑制效率。實(shí)驗(yàn)中分別構(gòu)建了4個表達(dá)單個siRNA的短發(fā)夾RNA(shRNA),8個同時表達(dá)兩個siRNA的長發(fā)夾RNA (1hRNA)和2個同時表達(dá)四個siRNA的雙長發(fā)夾RNA (dlhRNA) o最后得到了抑制效果最好的雙長發(fā)夾RNA表達(dá)原件dlhRNA-VGTE,并將dlhRNA表達(dá)原件導(dǎo)入FG12慢病毒載體。在TZM-bl細(xì)胞中,經(jīng)過持續(xù)加入FG12-VGTE病毒(MOI=0.04)可以完全抑制HIV-1病毒的復(fù)制。本文中還利用FG12-VGTE慢病毒構(gòu)建了表達(dá)dlhRNA-VGTE的VGTE-TZM-bl細(xì)胞系。VGTE-TZM-bl細(xì)胞可以抵抗HIV-1病毒的感染達(dá)10天之久。同時dlhRNA表達(dá)原件不會誘導(dǎo)細(xì)胞內(nèi)干擾素通路的激活。所以,多靶位RNAi有望被用作HIV-1的基因治療,為進(jìn)一步展開HIV-1的基因治療提供了參考依據(jù)。
[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.

【學(xué)位授予單位】：南開大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.91

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相關(guān)期刊論文 前4條

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本文編號：1701238