HIV-vif基因的RNA干扰试验研究

发布时间：2018-06-13 04:52

本文选题：人类免疫缺陷病毒(HIV) + 艾滋病　；参考：《复旦大学》2008年硕士论文

【摘要】： 随着人类免疫缺陷病毒(HIV)感染人数的增加,艾滋病的流行已经给国家经济和社会带来了巨大负担。但目前的抗病毒药物无法给患者提供彻底的治疗,因此更多的研究开始转变以往的治疗思路,探索新的更有效更经济的艾滋病治疗方法。RNAi(RNA interference,RNA干扰)研究的出现,为HIV的抑制及治疗提供了一种新的尝试。RNAi干扰效应是指双链RNA在生物细胞内对序列特异性基因转录和表达的抑制作用。RNAi的研究为控制HIV感染带来希望,通过SiRNA对病毒生活周期的多个水平作用,可显著抑制病毒的复制。目前已有研究报道了通过对HIV-1 gag,tat,rev和宿主CD4及CCR5受体进行RNA干扰,进而达到抑制HIV病毒复制的目的,并且研究取得了一定的进展,为抗HIV治疗开辟了一条新路。 HIV-1的特点之一是具有多个调节基因,这些基因可编码相应的调控蛋白,如Nef、Tat、Rev、Vif、Vpr、Vpx、Vpu等,它们在RNA转录、转录后加工、蛋白质翻译直到病毒颗粒从细胞膜释放的过程中发挥重要的调节作用。Vif蛋白被归于附属蛋白质,编码一个含有192个氨基酸,相对分子质量大约23000的胞质蛋白,vif基因比HIV-1基因组中env、gag等突变率高的基因相对保守。但10多年前就已经知道Vif是HIV-1感染所必须的调控蛋白,vif基因缺陷性(△vif)的HIV-1和SIV在体内无法复制,在体外虽可正常复制并产生病毒颗粒但其感染性比野生型降低近1000倍以上。vif缺陷性的病毒颗粒在形态学上也表现异常。在子弹头状病毒核心的电子密集物质明显减少,而核心与包膜之间的空间里电子密集物质明显增加,使核心形状呈多样化。另外,在感染细胞中的反转录过程以及病毒颗粒中的内源性反转录也是明显受损,因而不能感染靶细胞。有趣的是,vif缺陷性的病毒在一些肿瘤细胞系可以顺利的进行复制。根据△vif HIV-1是否能够在其内复制可将细胞分为“允许细胞”和“非允许细胞”。△vif HIV-1病毒可以进入非允许细胞中,但在反转录过程中或反转录之后到整合之前由于某种尚不清楚的机制导致感染终止。早期研究发现,除了感染性降低1000倍以上外,△vif HIV-1同野生型HIV-1相比较,二者在RNA结构及蛋白表达等方面均未发现任何差别。近期由于载脂蛋白B mRNA编辑酶催化多肽样蛋白3G(apolipoprotein B mRNAediting enzyme catalytic polypeptide like 3G,APOBEC3G)的发现以及对APOBEC3G与Vif相互作用机制的深入研究,使越来越多的研究者开始重视vif基因及其表达产物在HIV生命周期中的作用。经过科研人员数年的努力,现在RNA干扰的机制已经得到比较清楚的阐述。RNAi是一种进化上保守的抵御转基因或外来病毒侵犯的防御机制,是指内源性或外源性大约为21～23bp的核苷酸与靶基因的转录产物mRNA存在同源互补序列的双链RNA(double-stranded RNA,dsRNA)在细胞内特异地降解该mRNA,从而致使特异性的基因有效封闭的过程,是一种序列特异性的转录后基因沉默(post-transcriptional gene silencing,PTGS)。RNAi作为一种简单、快速、特异、高效、经济、效果可预测的技术,具有明显的优点,它比反义核酸技术优越,比基因敲除简单,具有很好的应用前景。具体可用于基因功能分析、研究信号传导通路的新途径、新药物的研究和开发、基因治疗、病毒感染治疗以及肿瘤治疗等各个方面。作为一种新的基因治疗药物,SiRNA可作为已有抗HIV病毒药物的辅助治疗,与其它抗病毒药物不会互相产生干扰,效果更好,相信随着对RNAi机制的进一步认识,这一技术能够成为抗病毒,特别是抗HIV感染的有力武器。因此本研究选择HIV-1 vif基因作为RNAi的靶目标,通过设计针对vif的特异性SiRNA对HIV病毒RNA进行干扰降解,进而达到降低或抑制病毒复制的目的。研究中我们成功的设计了3段vif特异性SiRNA,对转接入pEGFP-N1质粒中的vif基因进行干扰,并分别在核酸和蛋白水平进行了验证,结果证明了RNAi可以有效且特异性的下调Vif蛋白的表达。本课题的研究目的就是利用RNAi技术对HIV-1 vif基因的转录水平进行下调,达到抑制Vif蛋白表达的目的,为新的抗HIV治疗和预防研究提供理论基础。研究首先选择人胚肾293T(HEK 293T)细胞作为宿主细胞,EGFP融合蛋白作为报告基因,观察HIV-1 Vif蛋白在细胞内的表达情况,为后续的RNAi做好准备。第一部分先建立pEGFP-N1-vif融合质粒,进行HEK 293T细胞转染,荧光显微镜观察转染细胞中的转染效率,蛋白印迹免疫分析技术(Western blot)检测Vif蛋白的表达情况。结果显示,HIV-1 Vif可以在HEK 293T细胞中成功表达。采用体外转录法合成SiRNA,首先合成六段SiRNA(四段针对vif和一段阳性对照、一段阴性对照)的正反义链模板,体外进行反转录,得到长21bp的双链SiRNA,然后双链SiRNA分别与pEGFP-N1-vif融合质粒共转染HEK 293T细胞。荧光下观察绿色荧光,筛选出能抑制HIV-1 vif基因表达的有效SiRNA。验证转染成功后进行细胞抽提总RNA,进行实时定量RT-PCR(Real-time PCR),验证干扰效果,Western blot检测蛋白表达情况。结果显示,与阴性对照比较,有3条SiRNA可明显抑制vifmRNA的表达,S-SiRNA1、S-SiRNA2组和S-SiRNA3分别下降了约53%、60%和63%;同时结果显示蛋白表达水平也明显降低,3条SiRNA的蛋白条带均明显减弱。最终验证了RNAi可以对HIV-1 vif进行有效且特异性的下调表达。本实验成功的建立了哺乳动物细胞pEGFP-N1-HIV-1/vif表达系统,并得以验证,Vif蛋白可以在体外哺乳动物细胞中高水平表达。研究应用RNAi技术,对HIV-1 vif从转录和表达水平进行了沉默,并分别在核酸和蛋白水平进行了验证,这对于阻断病毒和宿主细胞结合及相互作用有重要启示,表明vif可以作为进一步研究抗HIV-1的潜力靶点,也为这一基因水平治疗手段的开展进行了积累。同时证实,RNAi作为一种简单、快速、特异、高效、经济、效果可预测的技术,具有明显的优点。但值得注意的是,本实验中的SiRNA也不能实现对HIV-1 vif的彻底敲除,干扰效率维持在50%左右。曾有研究报道,RNAi对HIV-1的干扰效率可以达到80%～95%左右。本实验考虑可能与实验操作和技术有一定的关系,也可能是vif基因的RNAi干扰效率较低造成的。但总之,RNAi研究的出现,为HIV的抑制及治疗提供了一种新的尝试。 RNAi技术的问世是生命科学发展史上的一大丰碑,若能长期稳定应用于抗病毒治疗,尤其是抗HIV治疗,无疑将造福人类。虽然目前RNAi技术在抗病毒的实验研究方面已取得可喜成绩,但由于诸多问题的存在,其应用于临床还有一段艰难漫长的道路。从理论和技术两个层面来解决RNAi的分子基础及其治疗应用问题任重而道远,因此对此项技术还需进一步的改进和创新。目前最好的艾滋病治疗方法,是用多种药物来干扰艾滋病毒生命周期中两种重要物质—反转录酶和蛋白酶—的作用,融合抑制剂与整合酶抑制剂也已成功应用于临床。但该方法并不能完全清除病毒,而且部分病人用药一段时间后,体内病毒产生了耐药性。因此,从HIV病毒生命周期的其它角度着手,开发新型药物,就显得十分重要。有研究者预期,新型疗法或许可以与现行的反转录酶抑制剂和蛋白酶抑制剂联合使用,适用于所有艾滋病毒感染者,而RNAi就是一种新的尝试和新的策略。
[Abstract]:With the increase of the number of human immunodeficiency virus (HIV) infection, the epidemic of AIDS has brought a huge burden to the economy and society. But the current antiviral drugs can not provide thorough treatment to the patients, so more research begins to change the previous treatment ideas and explore new and more efficient and more economical AIDS treatment. The emergence of the study of.RNAi (RNA interference, RNA interference) provides a new attempt on the inhibition and treatment of HIV for the inhibitory effect of.RNAi on the inhibitory effect of the double chain RNA on the transcription and expression of sequence specific genes in biological cells, which brings hope for controlling HIV infection, and the multiple levels of the life cycle of the virus through SiRNA. The role of the virus can significantly inhibit the replication of the virus. At present, it has been reported that through RNA interference on HIV-1 gag, tat, rev and host CD4 and CCR5 receptors, the purpose of inhibiting the replication of HIV virus is achieved, and some progress has been made in the study, which opens a new way for the anti HIV treatment.
One of the characteristics of HIV-1 is that there are multiple regulatory genes that can encode the corresponding regulatory proteins, such as Nef, Tat, Rev, Vif, Vpr, Vpx, Vpu and so on. They play an important regulatory role in RNA transcriptional, post transcriptional processing, protein translation until the release of virus particles from the cell membrane, and the.Vif protein is assigned to the accessory protein and encodes a protein. The vif gene, which contains 192 amino acids and relative molecular mass of about 23000, is relatively conservative than the genes with high mutation rates such as Env and gag in the HIV-1 genome. But more than 10 years ago, it has been known that Vif is a necessary regulatory protein for HIV-1 infection. The HIV-1 and SIV of the vif gene defect (delta VIF) can not be replicated in the body, although it can be recovered in vitro. The virus particles that produce and produce virus particles, but the virus particles whose infectivity is nearly 1000 times more than that of the wild type, are also abnormal in morphology. The electron dense substance in the core of the bullet head virus is obviously reduced and the electron dense substance in the space between the core and the envelope is significantly increased and the core shape is diversified. In addition, the.Vif virus core is varied. The reverse transcriptional process in the infected cells and the endogenous reverse transcription in the virus particles are also significantly impaired, and therefore cannot infect the target cells. Interestingly, the Vif defective virus can be successfully replicated in some tumor cell lines. If the Vif HIV-1 can be reproduced within it, the cells can be divided into "permissive" and "non" Allow cells ". Delta Vif HIV-1 virus can enter non permissible cells, but in the reverse transcription process or after reverse transcription to integration due to some unknown mechanism leading to infection termination. Early studies found that, in addition to 1000 times the infection reduction to the upper, delta Vif HIV-1 is compared with the wild type HIV-1, the two in RNA structure and protein In recent years, the discovery of the apolipoprotein B mRNA editing enzyme catalyzed polypeptide like protein 3G (apolipoprotein B mRNAediting enzyme catalytic polypeptide like 3G, APOBEC3G) and the in-depth study of the interaction mechanism between them and the interaction mechanism have made more and more researchers begin to attach importance to the genes and their genes. The role of the expression product in the HIV life cycle.
After several years of efforts by researchers, the mechanism of RNA interference has been clearly stated that.RNAi is an evolutionary conservative defense mechanism to resist transgene or alien virus invasion, which refers to the dual chain RNA of endogenous or exogenous nucleotide and target gene transfer product mRNA with a homologous complementary sequence of 21 to 23bp. (double-stranded RNA, dsRNA) is a simple, rapid, specific, efficient, efficient, efficient, efficient, economical, and predictable technique, which is a simple, rapid, specific, efficient, efficient, cost-effective, and predictable technique, which degrade the mRNA in the cell specifically, and causes the effective closure of specific genes. It is a sequence specific post transcriptional gene silencing (post-transcriptional gene silencing, PTGS).RNAi as a simple, rapid, specific, efficient, economical and predictable technique. It is superior to antisense nucleic acid technology and is more simple than gene knockout. It can be used in gene functional analysis, new pathway of signal transduction pathway, research and development of new drugs, gene therapy, virus infection treatment and tumor treatment. As a new gene therapy drug, SiRNA can be used as a new gene therapy drug. It is believed that this technology can become a powerful weapon for anti viral, especially anti HIV infection, as the adjuvant therapy of anti HIV virus drugs and other antiviral drugs do not interfere with each other. It is believed that with the further understanding of the RNAi mechanism, this technique can be a powerful weapon for anti viral, especially anti HIV infection. Therefore, this study chose the HIV-1 vif gene as the target of RNAi and designed for vi. F specific SiRNA interferes with HIV virus RNA, and then reduces or inhibits virus replication. In the study, we successfully designed 3 segments of Vif specific SiRNA to interfere with Vif genes transferred into pEGFP-N1 plasmids, and verify the nucleic acid and protein level respectively. The results show that RNAi can be effective and specific. The expression of Vif protein is downregulated by sex.
The purpose of this study is to reduce the transcriptional level of the HIV-1 vif gene by using RNAi technology to inhibit the expression of Vif protein, and to provide a theoretical basis for the new anti HIV treatment and prevention research. First, the study selected human embryonic kidney 293T (HEK 293T) cells as host cell, EGFP fusion protein as a reporter gene, and observed HIV-1 Vi. The expression of F protein in the cell was prepared for the subsequent RNAi. The first part first established the pEGFP-N1-vif fusion plasmid, transfected HEK 293T cells, and observed the transfection efficiency in the transfected cells by fluorescence microscopy. The expression of Vif protein was detected by Western blot immunoassay (Western blot). The results showed that HIV-1 Vif could be in HEK. 293T cells were successfully expressed. SiRNA was synthesized by in vitro transcription. First, six segments of SiRNA (four segments for Vif and a positive control, a negative control) were synthesized by reverse transcription, and a long 21bp double chain SiRNA was obtained in vitro. Then the double stranded SiRNA and pEGFP-N1-vif fusion plasmid co transfected HEK 293T cells. Detection of green fluorescence, screening effective SiRNA. to inhibit the expression of HIV-1 vif gene to verify the successful transfection of the total RNA, real-time quantitative RT-PCR (Real-time PCR), verify the interference effect, Western blot detection protein expression. The results show that, compared with negative control, 3 SiRNA can obviously inhibit vifmRNA expression, S-SiRNA1 The S-SiRNA2 group and S-SiRNA3 decreased by about 53%, 60% and 63%, respectively, and the results showed that the protein expression level was also significantly reduced, and the protein bands of 3 SiRNA were obviously weakened. Finally, it was proved that RNAi could be effectively and specifically down expression of HIV-1 Vif.
This experiment successfully established the pEGFP-N1-HIV-1/vif expression system of mammalian cells and proved that Vif protein could be expressed at high level in mammalian cells in vitro. RNAi technology was used to study the transcription and expression level of HIV-1 Vif, and the level of nucleic acid and protein was verified respectively, which was used to block the virus. The binding and interaction of host cells have important implications, indicating that Vif can be used as a potential target for further research on the potential of anti HIV-1 and also for the development of this gene level treatment. It is also proved that RNAi has obvious advantages as a simple, rapid, specific, efficient, economical and predictable Technology. In this experiment, SiRNA can not complete the complete knockout of HIV-1 Vif, and the interference efficiency is about 50%. The interference efficiency of RNAi to HIV-1 can reach about 80% to 95%. This experiment may have a certain relationship with the experimental operation and technology, and can also be caused by the low RNAi interference efficiency of the vif gene. In conclusion, the emergence of RNAi provides a new attempt for inhibition and treatment of HIV.
The advent of RNAi technology is a great monument in the history of life science. If it can be applied to antiviral therapy for a long time, especially anti HIV treatment, it will undoubtedly benefit mankind. Although RNAi technology has made gratifying achievements in the experimental research of antivirus, it has a long and difficult application in clinical due to the existence of many problems. The best way to treat RNAi is to interfere with the two important substances in the life cycle of HIV - reverse transcriptase and protease in the life cycle of HIV. The effects, fusion inhibitors and integrase inhibitors have also been successfully applied to the clinic. But this method does not completely remove the virus, and some patients have a drug resistance after a period of drug use. Therefore, starting new drugs from the other angles of the HIV virus life cycle is very important. The new therapy may be combined with the current reverse transcriptase inhibitors and protease inhibitors, suitable for all HIV infected people, and RNAi is a new attempt and a new strategy.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R512.91;R346

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