淡水微藻凝集素之純化、特性分析與免疫標定

Abstract

凝集素又稱為親醣蛋白，廣泛分佈在自然界之許多生物體內，由於其能與特殊醣類進行特異性之辨識與結合，是一種能與血球或細胞產生凝集現象之醣蛋白。本研究主要是針對淡水微細藻類之血液凝集活性進行調查並檢定，將44株淡水微細藻萃取液與人類正常A、B、O型與動物牛、羊、豬之紅血球進行凝集活性測試。 過去文獻指出萃取藻類時，許多因素諸如萃取溶劑、溫度、反應血球之來源等，均會造成萃取液凝集活性之改變。本實驗顯示以20% (V/V) 之酒精萃取藻類所得萃取液之凝集活性較高，而大部分藻類萃取液均能至少與一種以上之紅血球進行凝集作用。對人類紅血球所產生凝集活性較為顯著；而動物紅血球中，以對豬紅血球產生之凝集作用較為靈敏，其次是羊與牛。大部分藻類萃取液與經胰蛋白酶或木瓜酶前處理後之紅血球反應，均能提高凝集活性，其中以木瓜酶作用後之豬紅血球，所得反應效果最好。藻類萃取液儲存於-20℃下4個月後之凝集活性完全消失。另外，將藻類萃取液進行抗菌活性測試，結果顯示藻類萃取液能有效拮抗病原菌，尤其是對枯草桿菌（Bacillus subtilis）之抑制效果最顯著。此結果顯示淡水藻類具有凝集活性與抗菌活性物質，未來可應用於醫療或生藥研究與開發。 依血球藻類萃取液產生凝集反應結果，進行群落分析發現能產生四種分類距離差異大的次群落。以血球凝集之特性進行主成份分析結果，則可順利地將Chlorella與Chlamydomonas區分為兩大區塊。最特殊的是多變值分析中，豬血球被作為藻類群聚與分群的依據。有趣的是依凝集活性對小球藻進行分群或群聚，與使用DNA序列分析小球藻結果相似，顯示藻類凝集素可能與細胞遺傳演化有關。因此推測微藻凝集素之進化是透過其轉變凝集特異性。此外，利用多變異分析可以方便地簡化與深入研究微藻凝集活性的變異。 此外，我們針對小球藻Chlorella pyrenoidosa進行培養、收集、凍乾保存並萃取，將此藻類萃取液進行凝集素純化。藻類萃取液經飽和硫酸銨沈澱、透析後，分別以膠體過濾管柱Sephacryl S-200與DEAE-Sepharose 陰離子交換層析法純化後，得到小球藻凝集素（Chlorella pyrenoidosa haemagglutinin；簡稱CPH）。由膠體過濾估計CPH之分子量約為60 kDa，以SDS電泳預估結果顯示分子量為58 kDa，因此推測CPH為一個單次元蛋白質。醣類抑制凝集活性測試結果顯示，醣蛋白對CPH之凝集活性具有抑制作用，以yeast mannan最具效果，但單醣與雙醣則不具抑制效果。胺基酸組成分析結果顯示，CPH之主 要組成為甘胺酸（Gly）。以原子力顯微鏡於非液相中觀察小球藻C. pyrenoidosa形態，結果顯示細胞外觀呈圓形，但並非十分平滑，細胞直徑約計4~5μm。於雲母片中固定超過2小時，則細胞有崩解現象產生。以原子力顯微鏡觀測所純化之CPH蛋白質結果顯示，發現該蛋白外觀為短桿狀，蛋白質之大小60 × 40 nm之桿狀蛋白質。以免疫電顯定位CPH在小球藻細胞之位置，結果顯示CPH多分佈於細胞壁內側與細胞膜外側之間。

Haemagglutinins have been found in a wide range of organisms. They are capable of agglutinating erythrocytes and other normal or transformed cells, with specific binding characteristics for carbohydrates to produce unique biological activities. We analyzed the haemagglutinating activity of algal extracts from 44 species of freshwater microalgae against native and trypsin/papain - treated cow, pig, sheep, and human A-, B-, and O-type erythrocytes. Algal extracts obtained with aqueous ethanol exhibited higher haemagglutinating activity than those obtained with aqueous acetone. Most of the algal extracts agglutinated at least one of the erythrocyte types analyzed. Human erythrocytes were the most sensitive of the cell types analyzed. In the other species, the sensitivity of algal haemagglutinating activity for erythrocytes was pig > sheep > cow. Pre-treating erythrocytes with trypsin and papain improved the detection of most algal agglutinins and increased the haemagglutination titer; pre-treatment with papain was most effective for pig erythrocytes. Algal extracts stored at –20 oC for four months lost their haemagglutinating activity. Algal extracts were also assayed for antibiotic activity against food pathogen bacteria. We also found that microalgae exhibited strong antibacterial activity against food pathogen bacteria, especially against Bacillus subtilis. Our numerical taxonomy data showed that these microalgae might be grouped into several clusters according to their haemagglutinating activity. Cluster analysis generated four distinct subclusters of taxa, characterized by different specificities for antigens or carbohydrate receptors on the erythrocytes. Principal component analysis further separated the haemagglutination characteristics of Chlamydomonas from Chlorella on the first two components. Specificity for pig erythrocytes accounted for most of the clustering or grouping of algal taxa in multivariate analysis. However, clustering or grouping patterns of Chlorella species on haemagglutinating activity resembled to that on DNA sequences, revealing a possible genetic connection of agglutinins and their biochemical characteristics in algal cells. Agglutinins in microalgae might have evolved through change of specificity with genetic methods. Variability of haemagglutination reactions among the algae investigated is simplified and interpreted most easily using multivariate analysis. Microalgal Chlorella pyrenoidosa were cells cultured, harvested, lyophilized and kept at –20 °C until used for extraction. Crude protein extraction solution was obtained from the powder via several steps including sonicating, centrifuging, precipitating, and dialyzing. We used gel filtration with high resolution Sephacryl S-200 column followed by DEAE-Sepharose procedure for purifying our target hemagglutinin. The purified protein, designated CPH, was a monomeric protein with apparent Mw of 58 kDa on SDS-PAGE and 60 kDa on gel filtration. Hemagglutinating activity of CPH was inhibited with glycoprotein, yeast mannan especially. The amino acid composition was rich in glycine. C. pyrenoidosa was observed with atomic force microscope (AFM) in air condition. The results show that algal morphology was spherical and surface was not smooth. According to the two and tree dimension and detail the morphology of C. pyrenoidosa as spherical shaped with 4-5μm diameters. In opposition, the CPH from C. pyrenoidosa was 20 and 40 nm in width and length, respectively.