本文關(guān)鍵詞：家蠅蛹凝集素的分離純化及對(duì)鼠腹腔巨噬細(xì)胞免疫功能的影響　出處：《天津科技大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 家蠅蛹 凝集素 分離純化 免疫活性 巨噬細(xì)胞

【摘要】：家蠅獨(dú)特的免疫防御機(jī)制一直是國(guó)內(nèi)外研究的熱點(diǎn)問題之一,因此,對(duì)家蠅免疫活性的研究具有重要的意義。本文在前期研究的基礎(chǔ)上,通過Con A Sepharose4B親和層析、DEAE弱陰離子交換層析、超濾凍干等方法從家蠅蛹中分離純化出一種新型的家蠅蛹凝集素。通過凝血活性及鼠腹腔巨噬細(xì)胞增殖實(shí)驗(yàn)篩選出最佳組分MBL-1。SDS-PAGE電泳及高效液相色譜證明其為單一蛋白,分子量約為24kDa。 研究結(jié)果表明MBL-1蛋白含量為97.2%,糖含量為2.55%,且希夫試劑糖染色實(shí)驗(yàn)證實(shí)其含有糖鏈結(jié)構(gòu)。MBL-1對(duì)兔血紅細(xì)胞的凝集效價(jià)為256,其凝血活性在0-40℃和pH6.0-9.0范圍內(nèi)保持相對(duì)穩(wěn)定,活性可被D-甘露糖和D-葡萄糖所抑制。同時(shí),通過β-消除反應(yīng)、紅外光譜、原子力顯微鏡和蛋白N端測(cè)序儀對(duì)MBL-1的結(jié)構(gòu)進(jìn)行了初步探討,證明了MBL-1是一種存在N-糖苷鍵,直徑60-100nm的橢球狀蛋白單體,肽鏈N-端封閉。經(jīng)串聯(lián)質(zhì)譜分析MBL-1的肽段序列與來源為家蠅的谷胱甘肽硫轉(zhuǎn)移酶1(GST-θ)具有較高的相似性。 MTT實(shí)驗(yàn)表明MBL-1可促進(jìn)巨噬細(xì)胞增殖,且具有濃度依賴關(guān)系,最佳作用濃度為40μg/mL,最佳作用時(shí)間為48h。中性紅吞噬實(shí)驗(yàn)結(jié)果表明MBL-1可以增強(qiáng)巨噬細(xì)胞的吞噬活性,此外,在一定范圍內(nèi)MBL-1能夠增加巨噬細(xì)胞NO的表達(dá)量。掃描電鏡結(jié)果顯示MBL-1還具有激活巨噬細(xì)胞的作用。MBL-1對(duì)巨噬細(xì)胞細(xì)胞因子IL-6、IL-12、TNF-α的分泌有影響。Western blot結(jié)果表明MBL-1激活了鼠腹腔巨噬細(xì)胞的ERK通路,促進(jìn)了巨噬細(xì)胞的增殖,ERK通路對(duì)巨噬細(xì)胞細(xì)胞因子IL-6、IL-12、 TNF-α的產(chǎn)生也有所影響。綜上所述,家蠅蛹凝集素對(duì)鼠腹腔巨噬細(xì)胞具有明顯的免疫調(diào)節(jié)活性,為天然免疫增強(qiáng)劑的開發(fā)應(yīng)用提供了一定的理論參考依據(jù),具有重要意義。
[Abstract]:The unique immune defense mechanism of Musca domestica has been one of the hot issues at home and abroad, so it is of great significance to study the immune activity of housefly. The weak anion exchange chromatography was performed by Con A Sepharose4B affinity chromatography. A new type of agglutinin was isolated and purified from the pupae of housefly by ultrafiltration freeze-drying method. The best component MBL-1.SDS-PAGE electrophoresis was screened by coagulation activity and peritoneal macrophage proliferation test. And high performance liquid chromatography (HPLC) showed that it was a single protein. The molecular weight is about 24 kDa. The results showed that the content of MBL-1 protein and sugar was 97.2and 2.55% respectively. The agglutination titer of rabbit red blood cells was 256 by Schiff reagent sugar staining. Its clotting activity remained relatively stable in the range of 0-40 鈩，

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