Rab32及其相互作用蛋白的鉴定分析

发布时间：2018-05-24 14:44

本文选题：抗原递呈细胞 + 树突状细胞　；参考：《第三军医大学》2011年硕士论文

【摘要】：树突状细胞(dendritic cells,DC)是主要的一种抗原递呈细胞,目前认为DC细胞抗原递呈的方式有三种:一种是DC细胞吞噬外来抗原,并加工处理形成表位肽,与MHCⅡ类分子结合,从而激活CD4+T细胞;另外一种指DC细胞将内源性合成的蛋白处理与MHCⅠ类分子结合激活CD8+T细胞;Bevan提出了一种新的DC细胞递呈抗原的方式——交叉递呈(cross presentation),它是指抗原递呈细胞将外源性的抗原加工处理,负载到MHCⅠ类分子表面形成复合物,从而激活初始CD8+T细胞引起细胞免疫反应的过程。交叉递呈对于监视组织中的肿瘤以及清除那些不能感染抗原递呈细胞的病毒起着十分重要的作用。详细了解DC细胞交叉递呈的机制将有助于我们更好的找到抗肿瘤和抗病毒的途径,为肿瘤和病毒感染的免疫治疗提供新的线索。但是目前对于DC细胞交叉递呈的调节机制研究甚少在前面的研究中,我们下调了57种活化型Rab蛋白的表达量,发现包括Rab32在内的12种Rab蛋白与抗原交叉递呈有关。目前,Rab32蛋白在抗原交叉递呈中的作用机制研究未明。因此,为了了解Rab32在抗原交叉递呈当中的工作机制,我们利用蛋白质组学的方法对Rab32及其相互作用蛋白进行鉴定分析。细胞内的生理活动通常是通过多个蛋白间形成蛋白复合物而实现的,因此,要了解某种生理功能,就需要了解这种蛋白质与蛋白质之间的相互作用网络。由于经典的蛋白质组学研究方法—酵母双杂交,只能够研究两两蛋白质间的相互作用以及假阳性率过高,串联亲和纯化结合质谱技术的方法逐渐得到发展。目的:鉴定分析Rab32及其相互作用蛋白方法:1. Profinity eXact标签在真核生物系统当中的应用在对蛋白质的纯化当中,亲和标签得到了广泛的应用。相对于其他标签而言,Profinity eXact标签作为一种新发明的标签具有洗脱,结合通过同一标签完成,不需要额外的步骤去除目的蛋白上的标签残留。由于利用该标签纯化后,在目的蛋白上没有残留,因此,所得到的目的蛋白更符合本身的生物特性。但是,Profinity eXact标签在真核系统当中还没有得到应用。因此,为了验证Profinity eXact标签是否能够在真核系统中很好的工作。我们构建了Profinity eXact标签融合表达EGFP的真核表达载体。利用磷酸钙沉淀法,我们将真核表达载体转染293FT细胞,使融合蛋白在293FT细胞中表达。然后,我们利用Profinity eXact标签对融合蛋白进行了纯化,并且利用荧光检测和SDS-PAGE胶结合考染的方法对纯化效果进行了验证。 2.鉴定分析Rab32及其相互作用蛋白为了鉴定Rab32的相互作用蛋白,我们构建了慢病毒载体FUEHTagEGFP、FUEHTagEYFPRab32。随后,我们建立了稳定表达的细胞系DC2.4FUEHTagEGFP、DC2.4TagEYFPRab32。在纯化过程中,我们对构建的TAP纯化平台的纯化条件进行了优化,构建了稳定的TAP纯化平台。随后,我们利用TAP技术对Rab32进行纯化,并且利用SDS-PAGE结合银染的方法对纯化效果进行验证。在获得纯化样本后,我们对样本进行了质谱分析,得到了可能是Rab32相互作用蛋白的蛋白。串联亲和纯化(TAP)包括两次亲和纯化,每次纯化所使用的纯化标签不一样。相对于亲和纯化而言,串联亲和纯化提高了样本的特异性,降低了背景杂蛋白的含量。随着质谱技术的发展,串联亲和纯化结合质谱技术应用,能够更好,更精准的检测和鉴定分析蛋白质复合物。因此,我们选择串联亲和纯化结合质谱技术的方法鉴定分析Rab32蛋白及其相互作用蛋白。结论: 1)我们首先成功构建了Profinity eXact标签的真核表达载体。然后,我们将Profinity eXact标签的真核表达载体通过磷酸钙沉淀法转染293FT细胞。我们观察到,融合蛋白在293FT细胞中表达良好。在纯化过程中,我们发现纯化蛋白主要通过第一次洗脱从亲和层析柱上洗脱下来。随后,我们进行SDS-PAGE跑胶和考马斯亮蓝染色,结果目的蛋白清晰可见。因此,我们认为Profinity eXact标签在真核系统当中工作良好,能够获得良好的纯化效果。2)我们首先构建了串联亲和标签EH-Tag的慢病毒表达载体FUEHTagEGFP、FUEHTagEYFPRab32。然后,我们建立了稳定表达的细胞系DC2.4FUEHTagEGFP、DC2.4FUEHTagEYFPRab32。随后,我们发现在第一次纯化的过程中合适的洗涤次数为7次,0.01M NaN3是洗脱效率最高的洗脱液。在纯化过程中,我们发现经过两次结合,两次洗脱后,我们得到的蛋白质量较少,因此,为了减少目的蛋白的损失,我们在第二次纯化过程中只结合不洗脱。最后通过质谱分析,我们发现了4个与Rab32蛋白共同纯化出来的蛋白,分别是Rab38、PHB、PHB2和MTA70 我们得出结论: Profinity eXact标签在真核系统中能够有效的工作。我们利用质谱对纯化样本进行分析,发现了4个蛋白,其中Rab38、PHB、PHB2可能是Rab32的相互作用蛋白。
[Abstract]:Dendritic cells (DC) is one of the main antigen presenting cells. Currently, there are three types of antigen presenting DC cell antigens: one is that DC cells phagocytic external antigens, and processing and processing the epitope peptide to combine with MHC class II molecules to activate CD4+T cells; the other means that DC cells process endogenous synthetic proteins with M. HC class I molecules combine to activate CD8+T cells; Bevan has proposed a new way of DC cell presenting antigen, cross presentation (cross presentation), which refers to the processing of exogenous antigen by antigen presenting cells, which is loaded to the surface of MHC class I molecules to form complex, activating the initial CD8+T cells to cause cellular immune response. A detailed understanding of the mechanism of the cross delivery of DC cells will help us better find ways to resist cancer and antivirus, and provide new clues for the immunotherapy of cancer and disease. At present, there is little research on the regulation mechanism of DC cell cross delivery.
In the previous study, we downgraded the expression of 57 active Rab proteins, and found that 12 Rab proteins, including Rab32, were related to the cross presentation of antigen. At present, the mechanism of the action of Rab32 protein in the cross presentation of antigen is not clear. Therefore, in order to understand the working mechanism of Rab32 in the delivery of antigen forks, we use protein. Identification and analysis of Rab32 and its interacting proteins by omics.
The physiological activity in cells is usually achieved through the formation of protein complexes between multiple proteins. Therefore, to understand a certain physiological function, we need to understand the interaction network between proteins and proteins. The classical proteomics research method, yeast two hybrid, can only study the interaction between 22 proteins. With the high false positive rate, serial affinity purification and mass spectrometry technology have been gradually developed.
Objective: to identify and analyze Rab32 and its interacting proteins
Methods: the application of 1. Profinity eXact tag in eukaryotic system.
Affinity labels have been widely used in the purification of proteins. Relative to other labels, the Profinity eXact label is eluted as a newly invented label, combined with the same label, and does not require additional steps to remove the label residue on the target protein. There is no residue, so the target protein is more consistent with its biological characteristics. However, the Profinity eXact tag has not been applied in the eukaryotic system.
Therefore, in order to verify whether the Profinity eXact tag can work well in the eukaryotic system, we have constructed the eukaryotic expression vector of the Profinity eXact tag fusion expression EGFP. We use the calcium phosphate precipitation method to transfect the eukaryotic expression vector to 293FT cells to express the fusion protein in 293FT cells. Then, we use Profinity e. The Xact tag was used to purify the fusion protein, and the purification effect was verified by fluorescence detection and SDS-PAGE gel binding test.
2. identification and analysis of Rab32 and its interaction protein
In order to identify the interaction proteins of Rab32, we constructed the lentivirus vector FUEHTagEGFP, FUEHTagEYFPRab32. then, we established a stable expression of the cell line DC2.4FUEHTagEGFP. In the process of purification, we optimized the purified TAP platform for the construction of the TAP purification platform, and constructed a stable TAP purification platform. After that, we purify Rab32 by TAP technology and verify the purification effect by combining SDS-PAGE with silver staining. After obtaining the purified sample, we have analyzed the samples by mass spectrometry to obtain the protein that may be Rab32 interacting protein. Series affinity purification (TAP) includes two affinity purification, each purification is used. The purification labels are different. In relation to affinity purification, tandem affinity purification improves the specificity of the sample and reduces the content of the background clutter. With the development of the mass spectrometry technology, the tandem affinity purification combined with mass spectrometry can better, more accurately detect and identify protein complexes. Therefore, we choose series affinity. The Rab32 protein and its interaction proteins were identified by purification and mass spectrometry.
Conclusion:
1) we first successfully constructed the eukaryotic expression vector of the Profinity eXact tag. Then, we transfected the Profinity eXact tag eukaryotic expression vector with the calcium phosphate precipitation method to transfect the 293FT cells. We observed that the fusion protein was well expressed in the 293FT cells. In the purification process, we found that the purified protein was mainly washed by the first washing. It was removed from the affinity chromatography column. Then, we performed SDS-PAGE running glue and kommassie blue staining. The results were clearly visible. Therefore, we think that the Profinity eXact label works well in the eukaryotic system and can obtain a good purification effect.2.) we first constructed the Lentivirus Expression of tandem affinity label EH-Tag. The carrier FUEHTagEGFP, FUEHTagEYFPRab32. then, we established the stable expression cell line DC2.4FUEHTagEGFP. Then, DC2.4FUEHTagEYFPRab32., we found that the appropriate washing times were 7 times during the first purification process, and 0.01M NaN3 was the eluant of the highest elution efficiency. In the purification process, we found two times, two times. After elution, we got less protein, so in order to reduce the loss of the target protein, we only combined with the second purification process. Finally, we found 4 proteins that were purified together with Rab32 protein, namely, Rab38, PHB, PHB2 and MTA70 by mass spectrometry analysis.
We conclude that Profinity eXact tags can work effectively in the eukaryotic system. We use mass spectrometry to analyze the purified samples and find 4 proteins, of which Rab38, PHB, and PHB2 may be the interacting proteins of Rab32.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【共引文献】