臺灣博碩士論文加值系統

CD23是免疫球蛋白E的第二型受器(FcεRII)，它被證明在調控IgE所媒介的疾病上扮演著關鍵的角色。CD23也被證明會事先形成三聚體或多聚體，牽涉許多過敏方面的疾病。在另一方面，有其它的研究指出CD23會被膜表面相關的蛋白酶分解，進而導致釋放水溶性的CD23 (sCD23)。本論文研究的目標，我們使用fibritin當作一個建構的基序，來促使水溶性的CD23形成穩定的三聚合體，我們稱此建構好的CD23為fibritin-CD23(fCD23)，利用此fCD23可以幫助闡明小鼠CD23與免疫球蛋白E之間的相互關係。為了建構此fCD23，我們將T4噬菌體的fibritin基序結合到小鼠細胞膜外完整CD23的氨基端，接著我們在E coli.蛋白質表現系統下，成功地誘導出大量的fCD23，透過使用兩種不同的親和性管柱層析法來純化fCD23，以及利用西方式墨點法來偵測44-kDa的單聚體fCD23以及其交叉聯結的多聚合體。在IgE和fCD23的結合能力測試上，我們使用結合實驗以及ELISA來偵測fCD23的結合能力。更進一步的探討是否fCD23可以抑制IgE所媒介的發炎性媒介物的釋放，我們使用??-hexosaminidase釋放實驗來檢測fCD23抑制釋放的能力。總結而論，這些結果顯示出，我們所建構的fCD23具有抑制RBL-2H3細胞的去顆粒化作用。在未來我們可以持續研究fCD23的特性，用以建立一個較強效的IgE競爭性抑制劑。

CD23 is a type II immunoglobin E receptor (Fc?n?悐II), was been demonstrated that it has play a crucial role in regulating IgE-mediated diseases. It also has been shown that CD23 can preassociate trimeric or oligomeric form involving many allergic diseases. On the other hand, other reports indicated that CD23 was decayed by membrane related protease, leading to release soluble CD23 (sCD23). In this dissertation, we used fibritin as a construct motif to form a stable trimeric sCD23. It can be described as fibritin-CD23 (fCD23). Utilizing fCD23 can elucidate the correlation between mouse CD23 and IgE. To construct fCD23, T4 phage fibritin sequence was ligated to mouse CD23 N-terminal, and then we successful induced large amount of fCD23 by E. coli. protein expression system. Using two affinity chromatography columns to purify fCD23, and we detected 44-kDa monomer fCD23 and cross-linked oligomer fCD23 by western blot analysis. In IgE and CD23 binding ability test, we used binding assay and ELISA to detect fCD23 binding ability. To further investigate whether fCD23 can inhibit IgE-mediated inflammatory mediator release, we also used?n??-?{hexosaminidase release assay to examine the inhibitory effects of fCD23. Collectively, the results revealed that fCD23 can inhibit degranulation in RBL-2H3 cells. In the future, we will be studied fCD23 property, to establish strong competitive inhibitor for IgE.

中文摘要 I
Abstract II
致謝 III
目錄 IV
前言 1
Fc?悐?飽AIgE的高親和性受器(high affinity receptor) 4
Fc?悐II，IgE的低親和性受器(low affinity receptor) 4
1.1 發現及分佈 4
1.2 CD23之cDNA基因序列 7
1.3 結構 9
1.4 調節 12
1.5 多聚合體的性質 13
1.6 和IgE作用的位置 14
1.7 其他的配體 17
1.8 蛋白酶的分解 18
1.9 生物功能 19
1.10 研究目的 20
材料與方法 23
1. 建構fibritin-CD23 ( fCD23 )基因段及其選殖 24
1-1、fibritin聚合酶連鎖反應 ( Polymerase Chain reaction；PCR )： 24
1-2、CD23聚合酶連鎖反應： 25
1-3、利用DNA電泳確認PCR產物: 25
1-4、DNA純化 ( Gel Extraction )： 27
1-5、接合反應 ( TA - cloning )： 28
1-6、菌株轉型 ( Transformation )： 29
1-7、藍白篩選 ( Blue-white screening )： 17
1-8、用PCR篩選單一菌落 (PCR screening)： 17
1-9、利用LB培養液培養單一菌落 (single colony)： 19
1-10、小量萃取質體DNA ( mini-prep )： 19
1-11、限制酶處理，確認質體DNA ( Restriction Enzyme Digestion )： 20
1-12、菌種保存 ( Glycerol stock )： 20
1-13、大量萃取質體DNA ( maxi-prep )： 20
1-14、使用限制酶確認大量萃取的質體DNA： 21
1-15、接合反應pET 43.1a-CD23 (K/X) Ligation： 22
1-16、限制酶處理，確認接合後的pET 43.1a-CD23 (K/X)： 22
1-17、限制酶處理： 22
1-18、接合反應pET 43.1a-CD23(K/X)-fibritin (E/K) Ligation： 23
1-19、限制酶處理，確認接合後的pET 43.1a-CD23(K/X)-fibritin (E/K)： 23
1-20、DNA定序 ( sequencing )： 23
2 水溶性NusA-fCD23的蛋白質表現及親和性管柱(affinity column)純化 24
2-1、pET 43.1a-fCD23 ( E/K/X )轉型到E coli. (Origami B)： 24
2-2、以IPTG誘導重組蛋白表現 ( IPTG induction )： 24
2-3、蛋白質電泳膠分析 ( SDS-PAGE )： 25
2-4、用超音波震盪破菌 ( Sonication )： 26
2-5、利用His•Tag親和性管柱純化重組蛋白 ( Ni2+ column )： 26
2-6、濃縮以及透析 ( Dialysis )： 27
2-7、利用S-protein agarose親和性管柱以及Thrombin純化重組蛋白： 28
3 水溶性fCD23融合蛋白的西方式墨點法(Western blotting)分析 28
3-1、蛋白質轉漬 ( transfer )： 28
3-2、西方式墨點法 (western boltting)： 29
4 水溶性fCD23蛋白的ELISA 30
4-1、透析 ( dialysis )： 30
4-2、ELISA分析： 30
5 水溶性fCD23蛋白與IgE結合反應ELISA 31
fCD23-IgE ELISA分析： 31
6 化學交聯物質(EDC)反應分析 32
交叉連結反應 ( cross linking )： 32
7 RBL-2H3細胞培養 32
8 水溶性fCD23蛋白影響RBL-2H3細胞的??-hexosaminidase釋放分析 33
8-1、fCD23和IgE結合反應： 33
8-2、96孔盤細胞培養： 33
8-3、??-hexosaminidase釋放： 33
結果 35
1. 製備fibritin-CD23基因及其相關基因片段 35
2. 用E coli.表現融合蛋白 37
3. 融合蛋白fCD23的純化 41
4. 融合蛋白fCD23的ELISA以及和IgE結合能力的ELISA分析 45
5. 重組蛋白fCD23的活性分析(??-hexosaminidase release assay) 48
6. 重組蛋白fCD23的化學交叉連結(chemical crosslinking)分析 52
綜合討論 54
參考文獻 55
附錄 65


圖目錄

圖1. 比較人類和小鼠CD23胺基酸序列(50-53) 8
圖2. 人類和小鼠單體CD23的圖示(56) 11
圖3. CD23以三聚體型態結合IgE 16
圖4. 建構fCD23基因段 72
圖5. fibritin-CD23建構圖示 73
圖6. 用pET22b載體表現融合蛋白fCD23 76
圖7. 在不同的E coli.中，用pET43.1a載體表現融合蛋白fCD23 77
圖8. 在不同的溫度下，用E coli.(Origami B)菌株表現融合蛋白fCD23 78
圖9. 對照組的蛋白質表現 79
圖10. 細菌萃取液的水溶性蛋白質粗產物分析 81
圖11. 用親和性管柱純化水溶性融合蛋白fCD23 82
圖12. 確認水溶性蛋白質產物可以被Thrombin作用 83
圖13. 用親和性管柱純化水溶性對照組蛋白 84
圖14. 水溶性融合蛋白fCD23以及其和IgE結合的ELISA分析 87
圖15. 測定DNP-BSA刺激RBL-2H3細胞釋放??-hexosaminidase的條件 90
圖16. fCD23抑制RBL-2H3細胞釋放??-hexosaminidase的分析 93
圖17. fCD23可以藉由EDC交叉連結 95
表目錄

表1. 實驗菌株 23
表2. 載體 23
表3. 製備fibritin以及CD23基因段的引子 24

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