凝集素 (lectin) 是一種具有醣類結合專一性之蛋白或醣蛋白，且因具有醣類專一性，所以能使紅血球發生凝集現象，也同時具有許多生理功能。本研究目的是將胡蘿蔔食用肉根部分的植物凝集素根據carbohydrate-binding specificity of the carrot lectin中所採用之萃取條件，利用管柱層析法將胡蘿蔔凝集素分離純化。另外也用其他兩種不同條件pH值之buffer進行萃取，比較其凝集活性。
純化部分，由實驗結果得知，可在硫酸銨飽和度達45% - 95%區間內得到較高比活性之蛋白質。經由陰離子交換層析管柱進一步純化，當鹽度達65-100%時可沖提出之蛋白質具有凝集活性。再利用親和性管柱進行下一步純化。
而萃取條件比較方面，利用5%醋酸（pH 2.5）、PBS buffer (pH 7.4) 和carbonate - bicarbonate buffer (pH 10.5) 三種條件，對 40 g 胡蘿蔔食用的根進行萃取。由實驗結果顯示，以carbonate - bicarbonate buffer 萃取之萃取物可得到最大之比活性。隨後比較N-acetyl-D-glucosamine、D(+)-glucose、D(+)-mannose、D(+)-galactose、maltose、D(+)-glucosamine、D(-)-fructose、saccharose 八種醣類對萃取物之活性抑制情形。結果顯示，雖然這八種醣類皆無法對各種萃取物之活性產生特異性之抑制，或許能夠提供將來純化之胡蘿蔔凝集素進一步研究之方向。

Lectins are proteins which can agglutinate erythrocytes, not only can bind carbohydrates, also have some specific biological activities.
In this study we extracted carrot root lectin and used precipitation method and chromatography to purify carrot root lectin. The extraction was also carried out in different pH buffers, looking for the effect of pH on hemagglutinating activities.
For purification, we extracted the lection activity by following Carbohydrate-binding Specificity of the Carrot .Ammonium sulfate fraction at 45 - 95% recovered the highest specific activity. For ion exchanger column, buffer B at 65 - 100 %, could elute the agglutinin activity. Finally, the affinity column could further purify carrot root lectin.
For the extraction step, we chose two other pH buffers: PBS buffer (pH 7.4) and Carbonate - Bicarbonate buffer (pH 10.5), and compared the effect with 5% acetate (pH 2.5), hoping to find out distinct hemagglutinating activities and carbohydrate’s specificities.

謝誌	I
中文摘要	III
英文摘要	IV
目錄	V
圖表目錄	VIII
縮寫表	X
第一章、緒論	1
第二章、文獻探討	2
2.1 凝集素之介紹	2
2.2 植物凝集素之功能	4
2.3 凝集素之生理功能與應用	4
2.4胡蘿蔔凝集素之調查研究	5
第三章、實驗材料與方法	12
3.1實驗材料	12
3.2實驗藥品	12
3.2.1胡蘿蔔萃取與純化	12
3.2.2凝集活性及特性分析	12
3.2.3蛋白質濃度測定	13
3.2.4 SDS蛋白質電泳分析	13
3.3實驗儀器	14
3.4實驗方法	15
3.4.1胡蘿蔔凝集素萃取	15
3.4.1.1酸性(5% acetic acid pH 2.5 )萃取	15
3.4.1.2中性(PBS pH 7.4 )萃取	15
3.4.1.3鹼性(Carbonate-bicarbonate buffer pH 10.5 )萃取	16
3.4.2胡蘿蔔凝集素之純化	16
3.4.2.1胡蘿蔔萃取物之濃縮	16
3.4.2.2硫酸銨沉澱範圍劃分	17
3.4.2.3陰離子交換層析	17
3.4.2.4 親和性管柱層析	18
3.4.2.4.1 親和性管柱合成	18
3.4.2.4.2 D(+)-Glucosamine親和性管柱純化	18
3.4.3凝集活性測定與醣類抑制作用	19
3.4.3.1血液來源	19
3.4.3.2配製2%紅血球懸浮液	19
3.4.3.3凝集活性測定	19
3.4.3.4醣類抑制凝集活性測定	20
3.4.4蛋白質含量測定	20
3.4.5 SDS 蛋白質電泳分析	21
3.4.5.1鑄膠	21
3.4.5.2樣品處理	21
3.4.5.3電泳	21
3.4.5.4膠體染色	22
3.4.5.4.1 Coomassie Blue stain	22
3.4.5.4.1 Silver stain	22
3.4.6 MALDI – TOF Mass 樣品製備	22
第四章、結果與討論	32
4.1胡蘿蔔凝集素之萃取	32
4.1.1不同pH 緩衝溶液對胡蘿蔔凝集素之影響	32
4.1.2胡蘿蔔萃取物醣類抑制測定	32
4.2 胡蘿蔔萃取液之濃縮試驗	33
4.3 胡蘿蔔凝集素之硫酸銨沉澱劃分	34
4.4 Q SepharoseTM Fast Flow 離子交換層析	35
4.5 Epoxy-activated SepharoseTM 6B 親和性層析	36
4.6 SDS電泳分析	37
4.7 MALDI - TOF質譜分析	38
第五章、結論	54
第六章、參考文獻	56

圖表目錄
圖 2-1、部分凝集素(merolectins)、整體凝集素(holoectins)、
嵌合凝集素(chimerolectins)之結構示意圖 11
圖3-1、萃取流程圖 24
圖3-2、胡蘿蔔凝集素純化流程圖 25
圖3-3、硫酸銨沉澱範圍劃分流程圖 26
圖3-4、凝集活性檢測示意 27
圖4-1、胡蘿蔔凝集素之硫酸銨沉澱劃分 46
圖4-2、(a) 胡蘿蔔凝集素之Q SepharoseTM Fast Flow
離子交換層析 (b) 胡蘿蔔凝集素之Q SepharoseTM Fast Flow
離子交換層(overload) 47
圖4-3、胡蘿蔔凝集素之Q SepharoseTM Fast Flow 離子交換層析
圖 4-4、胡蘿蔔凝集素之 Epoxy-activated SepharoseTM 6B 48
親和性層析(Peak A) 49
圖4-5、(a)胡蘿蔔凝集素之 Epoxy-activated SepharoseTM 6B
親和性層析(Peak B) (b)利用KSCN 再次沖提 50
圖4-6、胡蘿蔔凝集素離子交換層析之SDS 膠體電泳圖(overload) 51
圖4-7、胡蘿蔔凝集素親和性層析之SDS 膠體電泳圖 52
圖4-8、胡蘿蔔凝集素親和性層析之SDS 膠體電泳圖
(Elute with KSCN) 53
圖4-9、Trypsin digestion 之Q SepharoseTM Fast Flow of carrot lectin
(overload) 40 kDa 之指紋圖譜 54
表 2-1、植物凝集素分類 7
表 2-2、具有抗蟲活性之植物凝集素 8
表 2-3、具有抗真菌活性之植物凝集素 9
表 2-4、凝集素之主要應用 10
表3-1、Stock dye solution 配置 28
表3-2、Dye-Reagent 配置 28
表3-3、12.5 % Separating gel 配置 28
表3-4、3.7 % Stacking gel 配置 28
表3-5、Tracking-dye 配置 28
表3-6、SDS running buffer 配置 29
表3-7、Stain buffer 配置 29
表3-8、Distain buffer 配置 29
表3-9、Silver solution 配置 29
表3-10、Developing solution 配置 29
表3-11、2X coupling buffer(pH 8.0)配置 29
表3-12、Acetate buffer(pH 4.0)配置 29
表3-13、Tris - HCl buffer(pH 8.0)配置 30
表3-14、Regenerated buffer 1 (pH 9.0)配置 30
表3-15、regenerated buffer 2 (pH 7.2)配置 30
表3-16、Carbonate-Bicarbonate (pH 10.5)配置 30
表3-17、Phosphate Buffered Saline 1M NaCl (pH 7.4)配置 30
表3-18、Phosphate Buffered Saline (pH 7.4)配置 30
表3-19、銀染退染劑配置 30
表3-20、Extraction 1 配置 31
表3-21、Extraction 2 配置 31
表4-1、不同pH 值之緩衝溶液活性比較 39
表4-2、醣類抑制5% acetic acid 胡蘿蔔萃取物之凝集活性試驗 40
表4-3、醣類抑制PBS 胡蘿蔔萃取物之凝集活性試驗 41
表4-4、醣類抑制Carbonate-bicarbonate buffer 胡蘿蔔萃取物之凝集活
性試驗 42
表4-5、醣類抑制5% acetic acid 胡蘿蔔萃取物之凝集活性試驗 43
表4-6、攪拌加壓過濾濃縮回收率比較表 44
表4-7、胡蘿蔔凝集素分離純化回收表 45

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