發(fā)布時間：2018-04-05 02:02

  本文選題：EF-Tu　切入點(diǎn)：OmpA　出處：《廈門大學(xué)》2008年碩士論文

【摘要】： 細(xì)菌延伸因子Tu(elongation factor Tu,EF-Tu)是一種高豐度蛋白,在蛋白質(zhì)生物合成的延伸過程中起到了關(guān)鍵性的作用。近來研究發(fā)現(xiàn)EF-Tu可以作為一種新的病原相關(guān)分子模式(Pathogen-associated molecular patterns,PAMPs)分子而被宿主識別。作為一個具有高度保守性的細(xì)菌蛋白質(zhì),它是如何被宿主細(xì)胞所特異性地識別并進(jìn)而引發(fā)宿主的哪些免疫應(yīng)答反應(yīng)。這無疑具有著非常重要的生物學(xué)意義。 本實(shí)驗(yàn)首先采用分子克隆和抗體制備技術(shù),以模式生物大腸桿菌K12為實(shí)驗(yàn)材料從其基因組中克隆出細(xì)菌延伸因子EF-Tu并制備其抗體。然后在本實(shí)驗(yàn)室之前研究EF-Tu多蛋白復(fù)合物的基礎(chǔ)上表達(dá)并純化了EF-Tu多蛋白復(fù)合物中OmpA、EF-Tu和OmpF三個蛋白組份。接著采用Western blotting和激光共聚焦證明了OmpA、EF-Tu和OmpF三個蛋白與人單核巨噬細(xì)胞THP-1有相互作用并被細(xì)胞膜受體識別而定位于細(xì)胞膜上。最后運(yùn)用酶聯(lián)反應(yīng)吸附試驗(yàn)技術(shù)對EF-Tu多蛋白復(fù)合物所引發(fā)人單核巨噬細(xì)胞THP-1細(xì)胞的免疫反應(yīng)進(jìn)行研究,發(fā)現(xiàn)EF-Tu多蛋白復(fù)合物中各蛋白組份可以明顯促使THP-1細(xì)胞細(xì)胞因子IL-Iβ、IL-8、IL-10、IL-12和TNF-α分泌量的增多。同時應(yīng)用流式細(xì)胞術(shù)發(fā)現(xiàn)EF-Tu多蛋白復(fù)合物中各蛋白組份可以顯著提高人單核巨噬細(xì)胞THP-1細(xì)胞的噬菌能力并具有劑量依賴效應(yīng)。 上述研究首次發(fā)現(xiàn)了EF-Tu多蛋白復(fù)合體可以作為一種新型病原相關(guān)分子模式通過細(xì)胞膜受體而被宿主免疫細(xì)胞所識別。闡明了其與人單核巨噬細(xì)胞THP-1細(xì)胞所引起的免疫學(xué)反應(yīng)。這些研究結(jié)果對深入闡明EF-Tu病原相關(guān)分子模式的生物學(xué)功能具有主要作用。
[Abstract]:Bacterial extension factor (Tu(elongation factor Tu-EF-TuS) is a high abundance protein, which plays a key role in the extension of protein biosynthesis.Recently, it has been found that EF-Tu can be recognized by the host as a new pathogen-associated molecular pattern pattern (PAMPs) molecule.As a highly conserved bacterial protein, how is it specifically recognized by host cells and then triggered by host immune responses.There is no doubt that this has very important biological significance.In this experiment, the bacterial extension factor (EF-Tu) was cloned from the genome of Escherichia coli K12 and its antibody was prepared by molecular cloning and antibody preparation techniques.Three protein components of EF-Tu polyprotein complex OmpAF-Tu and OmpF were expressed and purified on the basis of the previous study in our laboratory.Then, Western blotting and laser confocal technique were used to demonstrate that OMP EF-Tu and OmpF interact with human mononuclear macrophage THP-1 and are recognized by cell membrane receptor and located on the cell membrane.Finally, the immunoreaction of human mononuclear macrophage THP-1 cells induced by EF-Tu polyprotein complex was studied by enzyme linked reaction adsorption assay (Elisa).It was found that the protein components of EF-Tu polyprotein complex could significantly increase the secretion of IL-10 IL-12 and TNF- 偽 in the THP-1 cytokines IL-I 尾 -IL-8, IL-10, IL-12 and TNF- 偽.At the same time, it was found by flow cytometry that each protein component of EF-Tu polyprotein complex could significantly improve the bacteriophage ability of human mononuclear macrophages THP-1 cells and had a dose-dependent effect.These studies have shown for the first time that EF-Tu polyprotein complexes can be recognized by host immune cells as a novel pathogen-associated molecular pattern through cell membrane receptors.The immunological reaction induced by human mononuclear macrophage THP-1 cells was elucidated.These results play an important role in elucidating the biological functions of EF-Tu pathogen-associated molecular models.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Molecular Cloning and Expression of PoIR2,a Novel Gene Involved in Immune Response in Japanese Flounder (Paralichthys olivaceus)[J];Journal of Ocean University of China;2010年01期

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本文編號：1712681