豬頷下腺黏多醣蛋白之分離、純化與特性分析

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题名:	豬頷下腺黏多醣蛋白之分離、純化與特性分析 Purification and Characterization of Submaxillary Mucins from Porcine
作者:	陳俊諺
贡献者:	生物科技研究所
关键词:	豬頷下腺黏多醣蛋白 N-羥基乙醯神經胺酸免疫反應
日期:	2013-07
上传时间:	2013-09-12 14:45:44 (UTC+8)
摘要:	豬頷下腺的黏多醣蛋白是一種醣蛋白，由一個蛋白質核心連接短鏈寡糖側鏈組成。豬頷下腺黏多醣蛋白主要的唾液酸成分為 N-羥基乙醯神經胺(Neu5Gc)，但有少量唾液酸成分為 N-乙醯神經胺酸(Neu5Ac)。然而，因為在人體細胞中缺乏合成 N-羥基乙醯神經胺酸的酵素，正常人體組織缺乏 N-羥基乙醯神經胺酸。人類的腫瘤細胞表面被發現有 N-羥基乙醯神經胺酸出現，因為腫瘤細胞在快速生長時，因為乙醯神經胺酸供應不及，因此會利用由體外攝入的 N-羥基乙醯神經胺酸，因此細胞或組織中N-羥基乙醯神經胺酸的出現可以作為一種腫瘤的標誌。許多報告提到純化時需要使用昂貴儀器，例如超高速離心；在此研究利用不同的純化方式探討豬頷下腺黏多醣蛋白的製備，以尋找一種便利且便宜的方法。 SDS-PAGE 分析顯示豬頷下腺含有3種不同分子量的黏多醣蛋白，接著使用陰離子交換管柱(HiTrap DEAE FF)以快速液相層析儀(FPLC)進行純化，可以得到單一的豬頷下腺的黏多醣蛋白。純化後，豬頷下腺的蛋白質回收率為6.4%，唾液酸的含量為1.2%。以人類週邊血液單核球細胞進行免疫活性探討，發現豬頷下腺黏多醣蛋白具有刺激發炎反應的效果，顯示豬頷下腺的黏多醣蛋白具有調節免疫的能力，值得進一步探討。 Porcine submaxillary mucin(PSM)is a glycoprotein composed of a protein core and frequent, short oligosaccharide side chains. N-Glycolylneuraminic acid(Neu5Gc)is the predominant sialic acid in PSM, and only a small amount of N-acetylneuraminic acid(Neu5Ac)was present. The gene corresponding to Neu5Gc synthesis in Homo sapiens is a notable exception, since Neu5Gc is absent from normal human tissues. In human cancer, considering the null expression of Neu5Gc in human somatic cells, the appearance of Neu5Gc in some human tumors was undoubtedly found to be a cancer marker. Many papers was addressed to the purification of mucins from bovine, porcine and ovine, but expensive equipment was used, such as ultracentrifuge; therefore, in this study, different ways were tried to purify submaxillary mucins from porcine and a convenient and cheap method was found and established. SDS-PAGE analysis showed that PSM possessed three glycoproteins at bands of 50 kDa, 60 kDa and 70 kDa. After purified by anion chromatography, a protein of single band was obtained with molecular weight about 60 kDa. After purification steps, the recovery of the mucin protein is 6.4% and the content of sialic acid is 1.2%. In immunomodulation analysis, it was found PSM could stimulate the peripheral blood mononuclear cells (PBMC) to present inflammatory effect; that is, PSM possessed a certain ability to modulate the immune response, which is worth to further study PSM’s role and mechanism on immunomodulation.
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