本文選題：LTB + RAW　； 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：大腸桿菌不耐熱腸毒素B亞單位是一種非常有效的粘膜免疫佐劑,它能刺激機(jī)體產(chǎn)生強(qiáng)烈的粘膜和系統(tǒng)免疫應(yīng)答。LTB通過與細(xì)胞表面的GM1結(jié)合,不僅能增強(qiáng)機(jī)體對(duì)抗原的免疫應(yīng)答效應(yīng),還能夠改變粘膜免疫應(yīng)答的類型。LTB的免疫調(diào)節(jié)作用包括促進(jìn)Th1和Th2型細(xì)胞反應(yīng)的細(xì)胞因子的表達(dá),CD8+T細(xì)胞的凋亡和CD4+T細(xì)胞的增殖;使B細(xì)胞活化的MHC‖、ICAM-1、CD25、CD40分子表達(dá)增加,影響B(tài)細(xì)胞和巨噬細(xì)胞的抗原處理和遞呈過程。但在巨噬細(xì)胞的具體免疫機(jī)制尚不清楚,本文將在這方面進(jìn)行研究。目的:從LTB處理的RAW264.7細(xì)胞中,篩選與LTB有相互作用的蛋白質(zhì)分子,并通過生物信息學(xué)方法預(yù)測(cè)其與LTB佐劑活性的相關(guān)性;并利用分子生物學(xué)技術(shù)對(duì)它們的相互作用進(jìn)行驗(yàn)證,最終探索LTB作為免疫佐劑的分子機(jī)理。方法:(1)在大腸桿菌中誘導(dǎo)表達(dá)含His-tag的LTB融合蛋白,用鎳磁珠純化。(2)復(fù)性后的LTB融合蛋白去除內(nèi)毒素,然后處理RAW264.7細(xì)胞。(3)提取LTB處理后RAW264.7細(xì)胞總蛋白,用pull down的方法,分離與LTB有相互作用的蛋白分子。(4)PAGE檢測(cè)分離得到的蛋白分子,并收集該蛋白質(zhì)進(jìn)行質(zhì)譜鑒定。(5)運(yùn)用生物信息軟件從質(zhì)譜鑒定結(jié)果中篩選出可能的相互作用蛋白分子,并對(duì)其進(jìn)行功能性分析和信號(hào)通路分析,構(gòu)建出這些蛋白分子相互作用的網(wǎng)絡(luò)圖,篩選出有意義蛋白分子。(6)免疫熒光驗(yàn)證這些分子在細(xì)胞內(nèi)與LTB相互作用。(7)Western blot檢測(cè)下游分子的表達(dá)。結(jié)果:(1)通過pull down捕獲到與LTB有相互作用的蛋白分子,并通過質(zhì)譜鑒定了這些蛋白分子。(2)通過對(duì)這些蛋白分子進(jìn)行生物學(xué)分析后,篩選出junction plakoglobin、heat shock protein 1、carbamoyl-phosphate synthetase 1、vimentin等25種有統(tǒng)計(jì)學(xué)意義的蛋白分子。(3)通過對(duì)25種蛋白分子進(jìn)行功能及其信號(hào)通路分析(3)免疫熒光結(jié)果顯示,Vimentin在生理狀態(tài)下不能與LTB相互作用,而GM130在細(xì)胞內(nèi)與LTB有相互作用,表明LTB在細(xì)胞內(nèi)是以內(nèi)吞小泡的形式運(yùn)輸?shù)摹?4)Western blot檢測(cè)顯示,LTB處理RAW 264.7細(xì)胞12 h后,β-actin表達(dá)上調(diào),Hspd1表達(dá)無(wú)變化,表明LTB與Actb確實(shí)存在相互作用。結(jié)論:通過質(zhì)譜鑒定結(jié)果和免疫熒光實(shí)驗(yàn),我們可以推測(cè)LTB通過與免疫細(xì)胞表面GM1結(jié)合,在Keratin家族和Actb等細(xì)胞骨架相關(guān)蛋白分子的協(xié)助下內(nèi)吞,并以小泡的形式到達(dá)高爾基體,通過高爾基體的加工、轉(zhuǎn)運(yùn)到Jup處或以抗原的形式直接遞呈給T細(xì)胞,通過Jup激活TCF/LEF,促進(jìn)T細(xì)胞的活化、增殖與分化,以及相關(guān)細(xì)胞因子的分泌,達(dá)到促進(jìn)免疫應(yīng)答的作用。
[Abstract]:E. coli heat-labile enterotoxin B subunit is a very effective mucosal immune adjuvant that stimulates the production of strong mucosal and systemic immune responses. LTB binds to GM1 on the cell surface. It can not only enhance the immune response to antigen, but also change the type of mucosal immune response. LTB can promote the expression of cytokines of Th1 and Th2 type reaction and the apoptosis of CD8 T cells and the proliferation of CD4 T cells. The expression of ICAM-1 CD25 + CD40 in B cells was increased, which affected the process of antigen treatment and presentation of B cells and macrophages. However, the specific immune mechanism of macrophages is not clear, this paper will study this aspect. Objective: to screen protein molecules interacting with LTB from RAW264.7 cells treated with LTB, and to predict their correlation with LTB adjuvant activity by bioinformatics, and to verify their interaction by molecular biological techniques. Finally, the molecular mechanism of LTB as an immune adjuvant was explored. Methods the LTB fusion protein containing His-tag was induced and expressed in E. coli. The LTB fusion protein was purified by nickel beads. The endotoxin was removed by refolding LTB fusion protein. The total protein of RAW264.7 cells treated with LTB was extracted from RAW264.7 cells. The total protein of RAW264.7 cells was extracted by the method of pull down. Isolation of protein molecules with interaction with LTB. Page was used to detect the protein molecules, and the protein was collected for mass spectrometry identification. 5) the possible interacting protein molecules were screened by bioinformatics software from the results of mass spectrometry identification. Functional analysis and signal pathway analysis were performed to construct the network diagram of the interaction of these proteins. The significant protein molecules were screened out and immunofluorescence was used to detect the expression of the downstream molecules by LTB interaction. Western blot was used to detect the expression of these molecules. Results the protein molecules interacting with LTB were captured by pull down and identified by mass spectrometry. 25 protein molecules, such as junction plakglobin shock protein 1 carbamoyl-phosphate synthetase 1 vimentin and so on, were screened. The immunofluorescence results showed that Vimentin could not interact with LTB in physiological state by analyzing the function and signal pathway of 25 protein molecules. However, GM130 interacted with LTB in cells, indicating that LTB was transported in the form of endocytosis vesicles. Western blot analysis showed that 尾 -actin expression was up-regulated in RAW 264.7 cells after 12 h treatment, indicating that there was indeed interaction between LTB and Actb. Conclusion: based on the results of mass spectrometry and immunofluorescence assay, we can speculate that LTB can be ingested with the help of Keratin family and cytoskeleton related protein molecules such as Actb, and reach Golgi body in the form of vesicles by binding to GM1 on the surface of immune cells. Through the processing of Golgi body, it was transported to Jup or presented directly to T cells in the form of antigen. TCFR / LEF was activated by Jup, which promoted the activation, proliferation and differentiation of T cells, and the secretion of related cytokines, so as to promote the immune response.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號(hào)：1774716