Talin1片段化对HIV-1感染的影响及相关蛋白质组学研究

发布时间：2018-11-17 10:48

【摘要】：研究背景： 细胞骨架踝蛋白(talin1)参与HIV-1入侵和突触形成过程,并且能够抑制HIV-1感染,但具体机制不明。我们通过差异蛋白质组学的方法,发现talin1在未经HARRT治疗的HIV-1感染者PBMCs中降解成一个片段,经过质谱鉴定该片段位于talin1C末端,约38kDa,含有整合素(intergrin)及actin结合位点,且进一步研究发现该片段与病毒载量负相关。通过生物信息学手段对鉴定的差异蛋白进行相互作用分析,结果显示,talin1参与了HIV-1功能基因pol和vif调控的网络,可见talin1片段在HIV-1感染过程中发挥重要作用,研究talin1在HIV-1感染中发生片段化及其抑制HIV-1感染的机制,有助于寻找介导HIV-1一宿主细胞相互作用的关键分子,为抗HIV-1药物研究提供药物新靶点。此外,HIV-1病毒的潜伏可能与HIV-1的入侵,整合以及复制逆转录等多个过程相关,潜伏机制非常复杂。而在HIV-1的入侵,整合及逆转录等过程,细胞质膜蛋白质都发挥重要作用,研究HIV-1潜伏过程中细胞质膜蛋白质的表达变化能为发现HIV-1潜伏机制及发现HIV-1潜伏标志物提供一定的线索。 研究目的： 研究talin1片段化与HIV-1感染的相关性,探讨talin1影响HIV感染分子机制,筛选HIV潜伏细胞模型中的质膜标志蛋白质,为了解HIV-宿主细胞相互作用的分子机制提供帮助。 方法： 1、构建HIV假病毒感染Tzm-b1细胞模型,研究talin1片断化与HIV感染的时间依赖关系；构建凋亡细胞模型,检验talin1片段化与细胞凋亡的相关性； 2、不同疾病患者PBMCs样本中检验talin1片段化的表达； 3、通过电击导入talin1蛋白片段及过表达38kDa talin1片段研究该38kDa talin1对HIV-1感染的影响； 4、通过iRNA干扰构建talin1缺失株,研究talin1缺失对HIV-1感染的影响,并通过蛋白质组学的方法探讨可能参与talin1片段化过程的相互作用蛋白质。 5、提取HIV-1潜伏细胞模型及其对照细胞的细胞质膜蛋白质,通过二维凝胶电泳分离蛋白,使用ImageMaster2D Platinum6.0进行图像分析软件分析差异蛋白质点。 结果： 1、Talin1在HIV-1感染初期就发生片段化,并且talin1片段化与细胞凋亡没有直接相关性； 2、Talin1在HBV患者中也发生了片段化,并在体外MLV病毒感染细胞模型中也检测到talin1片段,且从感染初期就发生片段化； 3、点击导入talin1蛋白片段能够明显抑制HIV-1的感染；过表达38kDatalin1片段也能抑制HIV-1的感染； 4、Talin1缺失能够促进HIV-1感染,talin1头部50kDa的功能与C端38kDa的功能相反,它能促进HIV-1感染； 5、HIV-1潜伏细胞模型质膜蛋白质组学研究发现16个差异蛋白质点,通过鉴定后得到12个非冗余蛋白质,其中3个蛋白质在HIV-1潜伏细胞中表达下调,9个蛋白质在HIV-1潜伏细胞中不表达； 结论： 初步研究talin1片断化与HIV感染的关系：HIV感染导致talin1片断化,talin1片断具有抗HIV的活性；Talin1片断以及在HIV潜伏感染细胞模型中发现的APR3可能为潜在的抗病毒药物或者药物靶标。
[Abstract]:Study Background: The cytoskeleton ankle (talin1) is involved in the process of HIV-1 invasion and synapse formation and is capable of inhibiting HIV-1 infection, but specific mechanisms We found that talin1 was degraded into a fragment from PBMCs of the HIV-1 infected person without the HARRT, and the fragment was identified by mass spectrometry at the end of the tinin 1C, about 38kDa, containing the integrin and actin binding. site, and further study found that the fragment was negative with viral load The results showed that talin1 was involved in the control of the HIV-1 function gene pol and vif, and it was found that the talin1 fragment played an important role in the process of HIV-1 infection. The role of talin1 in the pathogenesis of HIV-1 infection and its inhibition of HIV-1 infection can help to find the key molecules to mediate the interaction of HIV-1 host cells and provide new drugs for anti-HIV-1 drug research. In addition, the latent mechanism of HIV-1 virus can be related to multiple processes of HIV-1 invasion, integration and reverse transcription, and the latent mechanism is very important. In the process of HIV-1 invasion, integration and reverse transcription, the cytoplasmic membrane protein plays an important role in the study of the expression of the cytoplasmic membrane protein in the HIV-1 latent process, which can be used to find the latent mechanism of HIV-1 and to find the latent mark of HIV-1. A clue. Objective: To study the relationship between talin1 fragmentation and HIV-1 infection, to study the molecular mechanism of talin1 on HIV infection, to screen the membrane marker proteins in the HIV latent cell model and to understand the interaction of HIV-host cells. molecular machine Methods: 1. The model of Tzm-b1 cells infected with HIV pseudovirus was constructed to study the time-dependent relationship between talin1 and HIV infection. the correlation between the fragmentation of n1 and the apoptosis of cells; 2. PBMCs of patients with different diseases sample the expression of the talin1 fragment was examined in. 3, the 38 kDa talin1 fragment was introduced by electric shock and the 38 kDa talin1 fragment was overexpressed the effect of kDa talin1 on HIV-1 infection; 4. Construction of the talin1 deletion via iRNA interference The effect of the deletion of talin1 on HIV-1 infection and the method of proteomics It is possible to take part in the interaction protein of the tinin1 fragmentation process. 5. Extract the cytoplasmic membrane proteins of the HIV-1 latent cell model and its control cells, isolate the proteins by two-dimensional gel electrophoresis, and use the ImageMaster2D P lat Image analysis software was used to analyze the difference protein points in inum6.0. Results: 1, Talin1 was infected with HIV-1. there was a fragmentation in the early stage, and the talin1 fragmentation was not directly related to cell apoptosis; 2, the Talin1 was also in the HBV patient Fragmentation occurs and in vitro ML the talin1 fragment was also detected in the V-infected cell model, and fragmentation occurred in the early stage of infection. the infection of HIV-1 is made; the overexpression of the 38kdatalin1 fragment can also inhibit the infection of HIV-1; 4, the absence of the Talin1 can promote the HIV-1 infection, The function of the talin1 head 50kDa was opposite to that of the C-terminal 38kDa, which can promote the HIV-1 infection; 5, the membrane proteome of the HIV-1 latent cell model was found to be 16 different protein points, and 12 non-redundant proteins were obtained by the identification. the mass, which The expression of 3 proteins in HIV-1 latent cells was down-regulated and 9 of the proteins were not expressed in HIV-1 latent cells. Conclusion: The relationship between talin1 fragmentation and HIV infection is studied in this paper.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91

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