本文選題：抗原遞呈細(xì)胞 + 樹突狀細(xì)胞��； 參考：《第三軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：樹突狀細(xì)胞(dendritic cells,DC)是主要的一種抗原遞呈細(xì)胞,目前認(rèn)為DC細(xì)胞抗原遞呈的方式有三種:一種是DC細(xì)胞吞噬外來(lái)抗原,并加工處理形成表位肽,與MHCⅡ類分子結(jié)合,從而激活CD4+T細(xì)胞;另外一種指DC細(xì)胞將內(nèi)源性合成的蛋白處理與MHCⅠ類分子結(jié)合激活CD8+T細(xì)胞;Bevan提出了一種新的DC細(xì)胞遞呈抗原的方式——交叉遞呈(cross presentation),它是指抗原遞呈細(xì)胞將外源性的抗原加工處理,負(fù)載到MHCⅠ類分子表面形成復(fù)合物,從而激活初始CD8+T細(xì)胞引起細(xì)胞免疫反應(yīng)的過(guò)程。交叉遞呈對(duì)于監(jiān)視組織中的腫瘤以及清除那些不能感染抗原遞呈細(xì)胞的病毒起著十分重要的作用。詳細(xì)了解DC細(xì)胞交叉遞呈的機(jī)制將有助于我們更好的找到抗腫瘤和抗病毒的途徑,為腫瘤和病毒感染的免疫治療提供新的線索。但是目前對(duì)于DC細(xì)胞交叉遞呈的調(diào)節(jié)機(jī)制研究甚少 在前面的研究中,我們下調(diào)了57種活化型Rab蛋白的表達(dá)量,發(fā)現(xiàn)包括Rab32在內(nèi)的12種Rab蛋白與抗原交叉遞呈有關(guān)。目前,Rab32蛋白在抗原交叉遞呈中的作用機(jī)制研究未明。因此,為了了解Rab32在抗原交叉遞呈當(dāng)中的工作機(jī)制,我們利用蛋白質(zhì)組學(xué)的方法對(duì)Rab32及其相互作用蛋白進(jìn)行鑒定分析。 細(xì)胞內(nèi)的生理活動(dòng)通常是通過(guò)多個(gè)蛋白間形成蛋白復(fù)合物而實(shí)現(xiàn)的,因此,要了解某種生理功能,就需要了解這種蛋白質(zhì)與蛋白質(zhì)之間的相互作用網(wǎng)絡(luò)。由于經(jīng)典的蛋白質(zhì)組學(xué)研究方法—酵母雙雜交,只能夠研究?jī)蓛傻鞍踪|(zhì)間的相互作用以及假陽(yáng)性率過(guò)高,串聯(lián)親和純化結(jié)合質(zhì)譜技術(shù)的方法逐漸得到發(fā)展。 目的:鑒定分析Rab32及其相互作用蛋白 方法:1. Profinity eXact標(biāo)簽在真核生物系統(tǒng)當(dāng)中的應(yīng)用 在對(duì)蛋白質(zhì)的純化當(dāng)中,親和標(biāo)簽得到了廣泛的應(yīng)用。相對(duì)于其他標(biāo)簽而言,Profinity eXact標(biāo)簽作為一種新發(fā)明的標(biāo)簽具有洗脫,結(jié)合通過(guò)同一標(biāo)簽完成,不需要額外的步驟去除目的蛋白上的標(biāo)簽殘留。由于利用該標(biāo)簽純化后,在目的蛋白上沒(méi)有殘留,因此,所得到的目的蛋白更符合本身的生物特性。但是,Profinity eXact標(biāo)簽在真核系統(tǒng)當(dāng)中還沒(méi)有得到應(yīng)用。 因此,為了驗(yàn)證Profinity eXact標(biāo)簽是否能夠在真核系統(tǒng)中很好的工作。我們構(gòu)建了Profinity eXact標(biāo)簽融合表達(dá)EGFP的真核表達(dá)載體。利用磷酸鈣沉淀法,我們將真核表達(dá)載體轉(zhuǎn)染293FT細(xì)胞,使融合蛋白在293FT細(xì)胞中表達(dá)。然后,我們利用Profinity eXact標(biāo)簽對(duì)融合蛋白進(jìn)行了純化,并且利用熒光檢測(cè)和SDS-PAGE膠結(jié)合考染的方法對(duì)純化效果進(jìn)行了驗(yàn)證。 2.鑒定分析Rab32及其相互作用蛋白 為了鑒定Rab32的相互作用蛋白,我們構(gòu)建了慢病毒載體FUEHTagEGFP、FUEHTagEYFPRab32。隨后,我們建立了穩(wěn)定表達(dá)的細(xì)胞系DC2.4FUEHTagEGFP、DC2.4TagEYFPRab32。在純化過(guò)程中,我們對(duì)構(gòu)建的TAP純化平臺(tái)的純化條件進(jìn)行了優(yōu)化,構(gòu)建了穩(wěn)定的TAP純化平臺(tái)。隨后,我們利用TAP技術(shù)對(duì)Rab32進(jìn)行純化,并且利用SDS-PAGE結(jié)合銀染的方法對(duì)純化效果進(jìn)行驗(yàn)證。在獲得純化樣本后,我們對(duì)樣本進(jìn)行了質(zhì)譜分析,得到了可能是Rab32相互作用蛋白的蛋白。串聯(lián)親和純化(TAP)包括兩次親和純化,每次純化所使用的純化標(biāo)簽不一樣。相對(duì)于親和純化而言,串聯(lián)親和純化提高了樣本的特異性,降低了背景雜蛋白的含量。隨著質(zhì)譜技術(shù)的發(fā)展,串聯(lián)親和純化結(jié)合質(zhì)譜技術(shù)應(yīng)用,能夠更好,更精準(zhǔn)的檢測(cè)和鑒定分析蛋白質(zhì)復(fù)合物。因此,我們選擇串聯(lián)親和純化結(jié)合質(zhì)譜技術(shù)的方法鑒定分析Rab32蛋白及其相互作用蛋白。 結(jié)論: 1)我們首先成功構(gòu)建了Profinity eXact標(biāo)簽的真核表達(dá)載體。然后,我們將Profinity eXact標(biāo)簽的真核表達(dá)載體通過(guò)磷酸鈣沉淀法轉(zhuǎn)染293FT細(xì)胞。我們觀察到,融合蛋白在293FT細(xì)胞中表達(dá)良好。在純化過(guò)程中,我們發(fā)現(xiàn)純化蛋白主要通過(guò)第一次洗脫從親和層析柱上洗脫下來(lái)。隨后,我們進(jìn)行SDS-PAGE跑膠和考馬斯亮藍(lán)染色,結(jié)果目的蛋白清晰可見。因此,我們認(rèn)為Profinity eXact標(biāo)簽在真核系統(tǒng)當(dāng)中工作良好,能夠獲得良好的純化效果。2)我們首先構(gòu)建了串聯(lián)親和標(biāo)簽EH-Tag的慢病毒表達(dá)載體FUEHTagEGFP、FUEHTagEYFPRab32。然后,我們建立了穩(wěn)定表達(dá)的細(xì)胞系DC2.4FUEHTagEGFP、DC2.4FUEHTagEYFPRab32。隨后,我們發(fā)現(xiàn)在第一次純化的過(guò)程中合適的洗滌次數(shù)為7次,0.01M NaN3是洗脫效率最高的洗脫液。在純化過(guò)程中,我們發(fā)現(xiàn)經(jīng)過(guò)兩次結(jié)合,兩次洗脫后,我們得到的蛋白質(zhì)量較少,因此,為了減少目的蛋白的損失,我們?cè)诘诙渭兓^(guò)程中只結(jié)合不洗脫。最后通過(guò)質(zhì)譜分析,我們發(fā)現(xiàn)了4個(gè)與Rab32蛋白共同純化出來(lái)的蛋白,分別是Rab38、PHB、PHB2和MTA70 我們得出結(jié)論: Profinity eXact標(biāo)簽在真核系統(tǒng)中能夠有效的工作。我們利用質(zhì)譜對(duì)純化樣本進(jìn)行分析,發(fā)現(xiàn)了4個(gè)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

【共引文獻(xiàn)】

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