小球藻中蛋白質分離前後物化性質差異之比較並分析在調節RAW 264.7巨噬細胞活性之角色

Physico-cheimical Properties of Proteins in Chlorella pyrenoidosa Before and After Separation and Analysis of Their Modulation on RAW 264.7 Macrophage Activation

蔡承順(Cheng-Shun Tsai)

指導教授 ： 程中玉

國立中正大學/理學院/化學暨生物化學系/碩士(2016年)

摘要

近年來，對於綠藻及其萃取物有許多研究。如何有效的將綠藻裡面有效的成分萃取並純化出來，並了解其性質組成和結構對於日後保健食品及藥物發展具有相當的幫助。因此本論文想從商業用的綠藻中分離出主要的蛋白質，做初步的結構分析並探討其對巨噬細胞活化的關係。
文中第一部分利用研磨、快速蛋白質分析儀搭配不同管柱和濃縮離心管一連串的方法將蛋白質分離純化並透過十二烷基硫酸鈉-聚丙烯醯胺膠體電泳不同染色法做判別。從綠藻粉末中成功的分離並純化出主要且最大量的15 KDa和30 KDa的蛋白質。在分離前後，利用不同光譜儀比較其差異性。在分離後在UV、FT-IR和拉曼上有明顯的蛋白質訊號峰存在，並推測15 KDa蛋白質序列可能含有色胺酸 (Trp)或纈氨酸 (Val)。而其主要的蛋白質二級結構為Random coil。
接著在細胞實驗上得知，加入純化後不同濃度的15 KDa蛋白質具有與LPS相同的功能去刺激巨噬細胞活化進而釋放NO，而30 KDa蛋白質卻沒有這樣的性質。之後再藉由XTT細胞毒性測試後，細胞存活率依然維持95%以上，證明此些蛋白質對細胞沒有毒性。

關鍵字

綠藻 ； 蛋白質萃取純化 ； LPS誘導巨噬細胞活化

並列摘要

In recent years, there are many research about green algae Chlorella pyrenoidosa and its extracts. How to extract bioactive proteins efficiently from green algae and to know their physic-chemical characteristics play an important role on drug develop and food industry. In this study, we extracted two major proteins from commercial green algae Chlorella pyrenoidosa. After purifying, we analyzed their qualitative properties and investigated their bioactivity on RAW 264.7 cells.
In this research, we used mortar and pestle with extract buffer, fast protein liquid chromatography with different columns and sodium dodecyl sulfate polyacrylamide gel electrophoresis with different staining ways to ensure our proteins were isolated. We successful separated and purified the two major proteins. (15KDa and 30KDa).Using spectra instrument to identify the difference before and after separation was conducted. We deduced that 15KDa protein might have Tryptophan or Valine on its sequence. And the major secondary structure is random coil in solution.
We found that the protein (15KDa) have an activity to induce RAW 264.7 cells for activation and promoted a reaction like LPS but the other protein 30KDa didn’t. In XTT assay there is no cytotoxicity to RAW 264.7 cells and cell viability is upper than 95%.

並列關鍵字

green algae ； protein extract and purify ； LPS induce RAW 264.7 macrophage activation

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