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,它能刺激機體產(chǎn)生強烈的粘膜和系統(tǒng)免疫應(yīng)答。LTB通過與細胞表面的GM1結(jié)合,不僅能增強機體對抗原的免疫應(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Th1和Th2型細胞反應(yīng)的細胞因子的表達,CD8+T細胞的凋亡和CD4+T細胞的增殖;使B細胞活化的MHC‖、ICAM-1、CD25、CD40分子表達增加,影響B(tài)細胞和巨噬細胞的抗原處理和遞呈過程。但在巨噬細胞的具體免疫機制尚不清楚,本文將在這方面進行研究。目的:從LTB處理的RAW264.7細胞中,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)對它們的相互作用進行驗證,最終探索LTB作為免疫佐劑的分子機理。方法:(1)在大腸桿菌中誘導(dǎo)表達含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細胞。(3)提取LTB處理后RAW264.7細胞總蛋白,用pull down的方法,分離與LTB有相互作用的蛋白分子。(4)PAGE檢測分離得到的蛋白分子,并收集該蛋白質(zhì)進行質(zhì)譜鑒定。(5)運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,并對其進行功能性分析和信號通路分析,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)免疫熒光驗證這些分子在細胞內(nèi)與LTB相互作用。(7)Western blot檢測下游分子的表達。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)通過對這些蛋白分子進行生物學(xué)分析后,篩選出junction plakoglobin、heat shock protein 1、carbamoyl-phosphate synthetase 1、vimentin等25種有統(tǒng)計學(xué)意義的蛋白分子。(3)通過對25種蛋白分子進行功能及其信號通路分析(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在細胞內(nèi)與LTB有相互作用,表明LTB在細胞內(nèi)是以內(nèi)吞小泡的形式運輸?shù)摹?4)Western blot檢測顯示,LTB處理RAW 264.7細胞12 h后,β-actin表達上調(diào),Hspd1表達無變化,表明LTB與Actb確實存在相互作用。結(jié)論:通過質(zhì)譜鑒定結(jié)果和免疫熒光實驗,我們可以推測LTB通過與免疫細胞表面GM1結(jié)合,在Keratin家族和Actb等細胞骨架相關(guān)蛋白分子的協(xié)助下內(nèi)吞,并以小泡的形式到達高爾基體,通過高爾基體的加工、轉(zhuǎn)運到Jup處或以抗原的形式直接遞呈給T細胞,通過Jup激活TCF/LEF,促進T細胞的活化、增殖與分化,以及相關(guān)細胞因子的分泌,達到促進免疫應(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)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R392

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相關(guān)期刊論文 前1條

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本文編號：1774716