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研究生:林芝安
論文名稱:1)利用DNA洗牌的方式促進GlcNAc2-epimerase的直接進化2)在自體免疫疾病中帶有高比率的抗中心體相關蛋白hNinein的自體抗體
論文名稱(外文):Direct evolution of GlcNAc 2-epimerase by DNA shuffling
指導教授:洪純隆洪純隆引用關係洪義人洪義人引用關係
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:102
中文關鍵詞:涎酸
外文關鍵詞:GlcNAc 2-epimeraseDNA shufflinghNineinautoimmune
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第一部分
Sialic acid ( NeuAc )因為分佈廣泛,結構上具多重功能及位在醣接合子的寡醣周邊位置,所以成為糖蛋白及糖脂質碳水鏈上為進行生物過程時的重要分子決定子,主要的功能包括細胞的黏滯性及訊息傳遞。而GlcNAc 2-epimerase對於催化Sialic acid biosynthetic pathway扮演一重要角色,但是在反應過程中須要耗費大量高成本的ATP,所以對於工業上量產siali1990年~1992年之間,豬、人、鼠的GlcNAc 2-epimerase基因相繼被完整clone出來,序列相亙比較發現其間存在有很多保留區域,而且其間的同源性也很高分別為人/鼠: 85.7 %、人/豬: 87.3 %、豬/鼠: 82.8 %。由於在這三種物種中,豬的GlcNAc 2-epimerase在sialic acid的生合成過程中,不須ATP的輔助也會有sialic acid 的生成,所以想藉由DNA shuffling 的技術把人、豬、鼠三種高同源性的 GlcNAc 2-epimerase進行改質期望得到能保有豬的酵素中不須ATP協助的特性,且酵素活性更佳的GlcNAc 2-epimerase。
目前己經從人、豬、鼠進行GlcNAc 2-epimerase的PCR放大,並利用重組DNA的方式把PCR放大的產物裝載到pET-14b,再轉型到E.coli中進行大量表達,並且得到大量的蛋白質產物,活性測試結果在E.coli中仍可保持其活性。另外引用” incremental truncation for the creation of hybrid enzymes ”的方法藉internal NcoI cutting site初步重組成功幾個重組的GlcNAc 2-peimerase的產物,活性測試結果有5~10 %的提升。
正利用DNase I進行DNA shuffling的工作,在未來的實驗中準備以GFPuv的基因作為一個篩選的marker,當作大量篩選的一個工具。以期在進行DNA洗牌重組之後能幫助大量篩選出能表達蛋白質且具有比原產物活性較高的重組基因,進而篩選出對ATP反應子需求量降低的GlcNAc 2-epimerase,而大幅降低反應成本,達到幫助工業化生產之目的。
c acid的製程中相對耗費很大的成本。
第二部分
一般來說,針對細胞內的抗原產生自體抗體是自體免疫疾病的一個主要特徵,只是這些自體抗體在疾病的致病原因上所扮演的角色並不是很清楚。中心體( centrosome )主要是負責有絲分裂紡錘體組織的胞器且在自體免疫的疾病中它們有時也是自體抗體的目標物。我們利用重組的中心體自體抗原,中心體專一性的抗體及免疫分析法去証實在自體免疫的疾病中明顯的會產生和中心體反應的抗體。
我們利用我們實驗室自己cloned出來的中心體相關蛋白hNinein以西方墨漬法去篩選約73個不同的自體免疫疾病的血清。且相同的這些血清再利用和ninein專一性的抗體做中心體的免疫螢光染色,可以用來區別當我們用自體免疫血清在做一般染色時,所產生的一些核的斑點和這中心體的差異。
從這些判斷標準的實驗結果來看,約有25-45 %的自體免疫的病人對中心體有自體反應的情況產生。相較於其他自體免疫疾病的自體抗原有蠻大比例的反應情況。我們挑出西方墨漬法中有反應的病人血清,在免疫螢光染色的觀察中也同樣可以看到病人血清和中心體反應的情況存在。在此,只有少數正常的健康人會有和中心體反應的情況產生。
這些結果顯示,針對中心體的自體抗體在自體免疫疾病中也是重要的會產生自體反應的組成物之一。所以,中心體自體抗原對於未來研究各種自體免疫疾病中自體抗體及慢性發炎反應之間的關係發展是很有用的。
PART IIn mammalian system, NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acetyl-D-glucosamine 2-epimerase ( GlcNAc 2-epimerase ). However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated. To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis the enzyme and its gene were isolated from porcine kidney cortex. Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney. Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein ( RnBP ) in porcine kidney ( Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y.1990 J.Biol.Chem. 265, 6556-6561 ) ( identity:99.6%in nucleotide sequence, 99% in amino acid sequence ).
Recently, N-Acetylneuraminate lyase ( aldolase ) and N-acyl-D- glucosamine 2-epimerase had been cloned and overexpressed in Escherichia coli. Simultaneous use of these two enzymes and feeding of appropriate amounts of pyruvate to the reaction to mixture made possible the high conversion of Neu5Ac from GlcNAc. However, GlcNAc 2-epimerase required ATP for activation. The cost of ATP is not applicable for industrial use. It is required to reengineer and search new source of GlcNAc 2-epimerase which exhibits no ATP requirement.
We have used reverse transcriptase polymerase chain reaction ( RT-PCR ) method to amplify cDNA from three species of human, rat and porcine. Then to subclone and recombinant the PCR products to pET-14b and transform to E.coli to overexpress N-acyl-D-glucosamine 2-epimerase protein. The enzyme activity maintain a status.
By the method of “the incremental truncation for the creation of hybrid enzymes”, we were used the internal NcoI cutting site to obtaine several clones, and the recombinant enzyme activity is shown increase 5~10%.
In the future, we will try to engineer these three GlcNAc 2-epimerase proteins by DNA shuffling combined GFPuv-fusion to find the more activity of the recombinant GlcNAc 2-epimerase than the wild type GlcNAc 2-epimerase for the highthroughput method by Industrial Technology Research Institute. Attempt to gain ATP-independent and low Km value GlcNAc 2-epimerase.
PART II
Autoantibodies to intracellular antigen are a hallmark of autoimmune diseases, although their role in disease pathogenesis is unclear. Centrosomes are organelles involved in the organization of the mitotic spindle and they are targets of autoantibodies in autoimmune diseases. We used recombinant centrosome autoantigens, centrosome-specific antibodies, and immunoassays to demonstrate that a significant proportion of autoimmune patients exhibited centrosome reactivity. One centrosome protein cloned in our laboratory were used to screen 73 autoimmune sera by Western blotting. The same sera were screened by immunofluorescence using centrosome-specific antibodies to distinguish centrosomes from nuclear speckles commonly stained by autoimmune diseases. Using these criteria, 25-45 % of autoimmune patients were autoreactive to centrosomes, a larger percentage than reacted with all other known autoimmune autoantigens. Only small percentage of normal individuals had centrosome reactivity. This results demonstrate that centrosome autoantibodies are a major component of autoreactivity in autoimmune diseases. Centrosome autoantigen may be useful in studying the development of autoantibodies and chronic inflammation in autoimmune diseases.
目錄
第一部份:
中文摘要 -------------------------------------1
英文摘要 -------------------------------------3
緒論 -------------------------------------5
材料與方法-------------------------------------14
實驗結果 -------------------------------------25
討論 -------------------------------------33
參考文獻 -------------------------------------36
圖表 -------------------------------------39
第二部份:
中文摘要 -------------------------------------53
英文摘要 -------------------------------------54
緒論 -------------------------------------55
材料與方法-------------------------------------62
實驗結果 -------------------------------------73
討論 -------------------------------------81
參考文獻 -------------------------------------85
圖表 -------------------------------------90
PART I
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PART II
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