本文選題：真菌免疫調(diào)節(jié)蛋白 + 菌落雜交篩選��； 參考：《上海交通大學(xué)》2012年碩士論文

【摘要】：真菌免疫調(diào)節(jié)蛋白(fugal immunomodulatory proteins,FIPs)是從高等擔(dān)子菌中分離出來的一類具有免疫調(diào)節(jié)活性的小分子蛋白質(zhì),目前已經(jīng)從多種高等擔(dān)子菌中分離得到天然蛋白質(zhì)以及克隆得到fip基因序列,真菌免疫調(diào)節(jié)蛋白具有廣泛的免疫調(diào)節(jié)特性,如促進(jìn)淋巴細(xì)胞的增殖,調(diào)控細(xì)胞周期,誘發(fā)細(xì)胞凋亡,影響細(xì)胞因子的表達(dá),活化細(xì)胞粘附分子,抗過敏反應(yīng)等等,具有巨大的蛋白質(zhì)藥物研究開發(fā)潛力。 本實驗將fip基因根據(jù)其來源分成兩組:屬內(nèi)組(fip-glu和fip-gsi)和屬間組(fip-glu、fip-fve和fip-vvo),利用DNA shuffling技術(shù)對這兩組基因在分子水平上進(jìn)行重組,構(gòu)建得到改組文庫,利用斑點雜交技術(shù)和特異性抗體對改組文庫共篩選426株屬內(nèi)重組菌株和412株屬間組菌株。經(jīng)過序列分析和蛋白誘導(dǎo)表達(dá)分析,屬內(nèi)組和屬間組基因重組蛋白完全符合改組模式,檢測重組蛋白的相對表達(dá)產(chǎn)量并篩選得到相對表達(dá)產(chǎn)量明顯提高的菌株;純化蛋白并進(jìn)行血細(xì)胞凝集實驗以及小鼠脾細(xì)胞因子誘導(dǎo)表達(dá)分析,實驗證明當(dāng)屬內(nèi)組FIP和屬間組FIP的濃度不低于0.20μg ml-1和1.56μg ml-1時,能夠凝集小鼠血細(xì)胞;重組FIP能夠誘導(dǎo)小鼠脾細(xì)胞因子interleukin(IL)-2, IL-3, IL-4, interferon(IFN)-γ, IL-2 receptor(IL-2R)和lymphotoxin(LT)基因的轉(zhuǎn)錄水平。 該項研究使利用基因工程和發(fā)酵技術(shù)進(jìn)行目的產(chǎn)物的產(chǎn)業(yè)化生產(chǎn)將逐漸成為可能,同時有助于探索該蛋白質(zhì)家族編碼基因的表達(dá)調(diào)控關(guān)系。
[Abstract]:Fungal immunomodulatory protein (immunomodulatory proteinsus) is a kind of small molecular protein with immunomodulatory activity isolated from higher basidiomycetes. At present, natural proteins have been isolated from a variety of higher basidiomycetes and fip gene sequences have been cloned. Fungal immunoregulatory proteins have a wide range of immunomodulatory properties, such as promoting the proliferation of lymphocytes, regulating cell cycle, inducing apoptosis, affecting the expression of cytokines, activating cell adhesion molecules, anti-allergic reactions and so on. It has great potential in protein drug research and development. In this study, fip genes were divided into two groups according to their origin: intra group fip-glu and fip-gsi. and intergeneric group, fip-glufifip-fve and fip-vvov. The two groups of genes were recombined at molecular level by shuffling technique, and the recombinant library was constructed. A total of 426 intrageneric recombinant strains and 412 intergeneric strains were screened by dot blot and specific antibody library. After sequence analysis and protein induced expression analysis, the recombinant proteins of intra and intergeneric groups were completely in accordance with the shuffling model. The relative expression yield of recombinant proteins was detected and the strains with higher relative expression yield were screened. The purified protein was purified by hemagglutination assay and the expression of spleen cytokines in mice was analyzed. The results showed that when the concentrations of intra and intergeneric FIP were not less than 0.20 渭 g ml-1 and 1.56 渭 g ml-1, the blood cells of mice could be agglutinated. Recombinant FIP can induce the transcription levels of interleukin-2, IL-3, IL-4, IFN- 緯, IL-2 receptor IL-2R) and lymphotoxin LTs genes in mice. This study will make it possible to use genetic engineering and fermentation technology to produce the target product, and it will be helpful to explore the expression and regulation of the protein family coding gene.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 蘇愷琪;真菌免疫調(diào)節(jié)蛋白表達(dá)調(diào)控研究[D];上海交通大學(xué);2013年

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本文編號：2017456