本文關鍵詞：ABO血型抗原的生物法合成及其與腸道菌關系的研究　出處：《山東大學》2015年博士論文　論文類型：學位論文

  更多相關文章： ABO血型 酶法合成 發(fā)酵法合成 細菌表面多糖 腸道菌群

【摘要】：糖類,廣泛存在于自然界的所有生物體中,不僅是不可或缺的生物體組成成分,也是生物體維持生命活動的主要能量來源。此外,糖類往往作為信息分子與其它分子相結合來發(fā)揮其重要的生物學功能。因此,以糖類為研究對象的糖生物學是了解生命過程和發(fā)展人類健康事業(yè)的重要科學,并日益被科學界和生物醫(yī)藥公司所重視。ABO血型系統(tǒng)是人類認識最早且最為普遍的血型分類系統(tǒng),可分為A、B、O和AB型四種,其血型抗原的本質是糖蛋白和糖脂,血型是由糖鏈末端的組成和結構決定的。組成ABO血型的單糖包括GlcNAc、Gal、GalNAc及fucose。目前,血型與疾病的關系已成為研究的熱點,現(xiàn)已知胃癌、胰腺癌、冠心病、高血壓、滲出性中耳炎和病毒性腦炎等疾病均在不同血型的人群中發(fā)病率不同,但相關研究仍處在病例統(tǒng)計分析水平,兩者之間相互作用的具體機理仍然不清楚。ABO血型抗原對于血型與人體健康的研究至關重要,因此,ABO血型糖抗原及其它生物活性糖類的制備對于其在生物醫(yī)學中的研究和應用顯得尤為重要。由于酶法合成寡糖具有高效化學和立體構型選擇性且經(jīng)濟省時,此法已趨向于替代傳統(tǒng)的化學合成方法。但由于Leloir型糖基轉移酶的底物糖核苷酸的數(shù)量有限且價格昂貴,限制了此法在大量合成生物活性寡糖方面的應用,嚴重阻礙了糖及糖綴合物在醫(yī)藥領域的研究和應用。糖核苷酸是單糖的活化形式,是由核苷一磷酸或核苷二磷酸與單糖的異頭碳連接形成的。糖核苷酸主要作為糖基轉移酶的底物發(fā)揮作用,不僅可以作為糖基供體來研究糖基轉移酶的生化性質,還可以通過對糖核苷酸中的單糖進行修飾從而形成糖基轉移酶抑制劑或將單糖衍生物添加到糖綴合物中來標記糖鏈或研究糖類的代謝途徑。生物酶法和微生物發(fā)酵法都有其各自的優(yōu)勢,均是有潛力實現(xiàn)糖核苷酸工業(yè)化生產(chǎn)的方法。腸道菌群與宿主之間是互利共生的,是人體不可分割的一個重要組成部分,相當于人體的一個重要“功能器官”,在宿主的代謝、免疫調(diào)控、防御、大腦發(fā)育以及人類行為等多方面都發(fā)揮著不可替代的作用。腸道菌群紊亂也與多種疾病的發(fā)生發(fā)展密切相關。在腸道菌群與宿主共同進化的過程中,來自宿主的遺傳因素、生活環(huán)境、飲食以及社會行為等內(nèi)在或外在因素都會影響腸道菌群的組成和結構。腸道菌表面多糖有脂多糖、莢膜多糖和胞外多糖,其中很多糖結構由于與人類ABO血型抗原相似而具有血型物質活性,能與ABO天然抗體發(fā)生免疫反應。因此探究ABO血型對腸道菌群組成的影響,不僅可以解釋血型與疾病易感性之間的關聯(lián),又可為ABO天然抗體起源的假說提供一個有力的佐證。本論文的目標之一是簡化UDP-GlcNAc的酶法合成并通過小規(guī)模制備得到其純品。UDP-GlcNAc是GlcNAc轉移酶催化的糖基化反應發(fā)生的先決條件。本課題組之前已用來自長雙歧桿菌的GlcNAc1位激酶(NahK)和來自大腸桿菌的截短的核苷酸焦磷酸化酶(truncated GlmU)合成了 UDP-GlcNAc,這里我們將這兩種酶進行了融合表達并摸索了融合酶的最適反應條件為pH 8.0,溫度40℃。之后,以GlcNAc, ATP和UTP為底物,用融合酶進行一步法反應生成了UDP-GlcNAc,產(chǎn)率為88%。反應液經(jīng)過堿性磷酸酶消化降解副產(chǎn)物后再經(jīng)過分子篩分離純化。最終總產(chǎn)率可達77%,得到的產(chǎn)品的純度可達90%以上。含有fucose的生物分子擁有多種生物學功能,而能夠催化其合成的糖基轉移酶的必要糖基供體底物為GDP-Fuc。在本論文的第三章中,我們將來自于脆弱擬桿菌的合成GDP-Fuc的補救途徑引入了大腸桿菌中。此外,由于GTP是GDP-Fuc生物合成的一個重要底物,我們過表達了 GTP生物合成補救途徑中的一些酶來增強它的合成。對加強表達的酶進行不同的組合,形成了不同的工程菌株,并通過搖瓶發(fā)酵比較了它們生成產(chǎn)物的能力。其中同時表達Fkp, Gpt, Gmk和Ndk的菌株產(chǎn)量最高,每克干重細菌內(nèi)含有4.6±0.22μmol,是只表達Fkp菌株的4.2倍。通過分批補料發(fā)酵,GDP-Fuc的產(chǎn)量進一步提高到6.6±0.14 μmol/g。與利用從頭合成途徑發(fā)酵生產(chǎn)GDP-Fuc相比,此方法不僅產(chǎn)量更高而且可以用來合成在fucose殘基上有化學修飾的GDP-Fuc類似物。影響腸道菌群組成的因素多種多樣,某些細菌的表面多糖具有ABO血型抗原活性,那么血型對腸道菌群組成的影響引起了我們的關注。本論文的第四章分析了提取于不同血型的171個個體的腸道菌群16SrDNA序列。不同血型人群的腸道菌群組成基本相似,但在不同的分類水平上存在一些差別。擬桿菌門、互養(yǎng)菌門以及柔膜菌門在不同血型人群中有差別,而其它門均無顯著性差異。在各分組間α多樣性在A型血人群的腸道菌群中最低,此外我們還發(fā)現(xiàn)A型和AB型人群的腸道菌中有益菌的數(shù)量比其它血型少。為了進一步探索腸道菌群適應宿主選擇壓的機制,我們進一步檢測了不同組的腸道菌群的血型抗原活性,發(fā)現(xiàn)A型血人群的腸道菌群有更高的A抗原活性,B型血人群的腸道菌群有更高的B抗原活性,而O型和AB型血人群的腸道菌群擁有相同的A和B抗原活性。這些結果說明ABO血型的確能夠影響腸道菌群的組成,細菌表面多糖的血型抗原活性不同是造成這種現(xiàn)象的原因之一,這就為血型與疾病易感性的機制研究指明了一個新的方向。總之,本學位論文利用酶法和微生物發(fā)酵法合成了兩種糖核苷酸。通過構建融合酶簡化了 UDP-GlcNAc的酶法合成,對大腸桿菌進行了代謝工程改造,獲得了高效合成GDP-Fuc的工程菌株。確定了 ABO血型對腸道菌群組成的影響并從細菌表面多糖與血型抗原的關系入手,初步解釋了血型與腸道菌群間的相互關系。
[Abstract]:Sugar, all organisms widely exist in nature, is not only the essential components of the organism, is the main energy source for organisms to maintain life activities. In addition, sugar is often used as signaling molecules and other molecules combine to play its important biological functions. Therefore, the carbohydrate sugar biology research object is important for understanding life science the process and development of the cause of human health, and increasingly by the scientific community and bio pharmaceutical companies pay more attention to the.ABO blood group system is the human understanding of the earliest and the most common type classification system, can be divided into A, B, O and AB four, its essence is antigen glycoproteins and glycolipids, blood type is determined by the the composition and structure of sugar chain ends. The monosaccharide composition of ABO blood group including GlcNAc, Gal, GalNAc and fucose. at present, the relationship between blood type and disease has become a hot research, it is known that gastric cancer, pancreatic Cancer, coronary heart disease, hypertension, exudative otitis media and viral encephalitis and other diseases in different type of incidence, but the related research is still in the case of statistical analysis, the specific mechanism of the interaction between them is still not clear.ABO antigen for the study of blood type and human health is essential, therefore, ABO blood group carbohydrate antigen and other bioactive carbohydrates were prepared for its research and application in biomedicine is particularly important. The enzymatic synthesis of oligosaccharides with high chemical and stereo selectivity and time saving, which has tended to replace the traditional chemical synthesis method. But due to the Leloir type glycosyltransferase substrate sugar nucleotide number limited and expensive, limiting the application of this method in a large number of synthetic oligosaccharides, the research has seriously hindered the sugar and sugar conjugates in the field of Medicine And the application of sugar nucleotide activated monosaccharides., is formed by the nucleoside monophosphate or nucleoside two phosphoric acid and monosaccharide anomeric connection. The nucleotide sugar mainly as glycosyltransferase substrates play a role in biochemical nature not only can be used as glycosyl donor of glycosyltransferases, carbohydrate can also be transferase inhibitors or metabolism of monosaccharide derivatives added to glycoconjugates in the labeled sugar chain or research sugar formed by modifying the monosaccharide sugar nucleotide in it. Enzymatic and microbial fermentation method has its own advantages, there was a realization method of industrial production potential. Sugar nucleotides between intestinal bacteria group and host is symbiotic, is an important part of the human body can not be split, one of the important functions of the organ "is equivalent to the human body, in host metabolism, immune regulation, defense, brain development in Human behavior and other aspects have played an irreplaceable role. The intestinal flora disturbance is closely related with the occurrence and development of many diseases. In the process of intestinal microflora and host coevolution in genetic factors from the host, living environment, diet and social behavior of internal or external factors will affect the composition and the structure of intestinal flora. Bacterial surface polysaccharides of lipopolysaccharide, capsular polysaccharide and extracellular polysaccharide, many sugar structure due to antigen and ABO blood type and blood group substances with similar activity, can produce immune reaction with ABO. The research of natural antibody ABO type on gut microbiota, not only can explain the association between blood type and disease susceptibility, but also for the origin of ABO natural antibody hypothesis provides a powerful support. One of the goals of this thesis is the synthesis of simplified UDP-GlcNAc method and enzyme by small scale system The prepared pure.UDP-GlcNAc is a prerequisite for glycosylation reaction catalyzed by GlcNAc transferase. Since the Bifidobacterium longum GlcNAc1 kinase has been used before the research group (NahK) and nucleotide pyrophosphorylase from Escherichia coli truncated (truncated GlmU) UDP-GlcNAc was synthesized, here we refer to the two enzyme the fusion expression and exploration of the fusion enzyme the optimum reaction conditions of pH 8, temperature 40 degrees. Then, using GlcNAc, ATP and UTP as the substrate, the formation of UDP-GlcNAc by fusion enzyme one-step reaction. The yield of 88%. reaction solution after alkaline phosphatase digestion degradation by-products after separation and purification of molecular sieve. The final total yield was 77%, the purity of the product is more than 90%. Biological molecules containing fucose has a variety of biological functions, and can catalyze the synthesis of glycosyl necessary glycosyltransferase donor substrate GDP-Fuc. in the third chapter, we come from the synthesis of GDP-Fuc fragilis the remedy into Escherichia coli. In addition, because GTP is an important substrate of GDP-Fuc biosynthesis, we have expressed some enzyme of GTP biosynthesis in the salvage pathway to enhance its synthesis by different combinations of. To strengthen the expression of the enzyme, the formation of the engineering strains, and to compare their ability to generate the products through fermentation. At the same time the expression of Fkp, Gpt, Gmk and Ndk were the highest yield per gram dry weight in bacteria containing 4.6 + 0.22 mol, is 4.2 times the only expression of Fkp strains by batch. Fed batch fermentation, the yield of GDP-Fuc further increased to 6.6 + 0.14 mol/g. compared with the use of de novo synthesis of fermentative production of GDP-Fuc, this method is not only higher yield and can be used to synthesize the fucose residues in a chemical modification GDP-Fuc analogs. Many factors influence the composition of intestinal microflora diversity, surface polysaccharides of some bacteria with ABO antigen activity, then blood group on gut microbiota attracted our attention. The fourth chapter analyzes the extracted from different blood group of 171 individuals of intestinal flora intestinal 16SrDNA sequence. The composition of flora of different blood groups are similar, but there are some differences in the classification of different levels. The Bacteroidetes, syntrophic bacteria and bacterial membrane soft door has the difference in the different blood groups, and the other was no significant difference in all groups. The diversity of intestinal flora in type A the blood in the lowest, we also found that the number of bacteria of intestinal bacteria A and AB groups than in the other group. In order to further explore the mechanism of intestinal flora to host selection pressure, we further examined the different groups The intestinal microflora of blood group antigen activity, A found that people with blood type A antigen activity of the intestinal flora in higher intestinal flora, people with type B blood B antigen have high activity, and the intestinal flora of type O and type AB blood groups A and B have the same antigen activity. These the results indicated that ABO blood type can influence the composition of the intestinal flora, antigen active bacterial surface polysaccharides is one of the reasons for this phenomenon, it is pointed out a new direction mechanism of blood type and disease susceptibility. In a word, in this thesis, two kinds of sugar nucleotide synthesis by enzyme method and microbial fermentation. By constructing a simplified synthetic fusion enzyme UDP-GlcNAc enzyme method, the metabolic engineering of Escherichia coli, the engineering strain for efficient synthesis of GDP-Fuc. The ABO blood group of gut microbiota and from the bacterial surface polysaccharides The relationship between the blood group and the intestinal microflora is explained with the relationship between the blood type and the blood type.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R457.11;R37

【相似文獻】

相關期刊論文 前10條

1 Jinwen Shi;Liejin Guo;;ABO_3-based photocatalysts for water splitting[J];Progress in Natural Science:Materials International;2012年06期

2 王怡明;;ABO血型多態(tài)性的分子基礎[J];國外醫(yī)學.輸血及血液學分冊;1991年05期

3 李安玲,梁芳,王淑燕,陳秋英,劉彥麗;臨汾地區(qū)獻血人群ABO抗原檢測與基因分布[J];山西師范大學學報(自然科學版);2000年03期

4 曹瓊,周華友,劉澤澎,饒軍華,劉曉明,章陽培,蘭炯采;華南獼猴紅細胞ABO血型研究[J];中國比較醫(yī)學雜志;2003年02期

5 毛慧;濟南市漢族人群ABO及RhD血型分布調(diào)查[J];臨床輸血與檢驗;2004年01期

6 宋斐 ,張陽;血型的ABO[J];小學科技;2004年07期

7 Bruce R ,郝巨為;人類ABO血型系統(tǒng)的生化遺傳學[J];國外醫(yī)學.遺傳學分冊;1983年05期

8 楊子元;西雙版納州基諾族、本族ABO血型調(diào)查[J];遺傳;1988年04期

9 郭建;;根據(jù)ABO血型能否判定出親生父母?[J];生物學雜志;1992年02期

10 陳峰;陳騰;閻春霞;黨永輝;穆豪放;于曉光;張博;鄧亞軍;;運用多重PCR-直接測序法檢測ABO基因型及其遺傳多態(tài)性[J];遺傳;2008年06期

相關會議論文 前10條

1 康利芳;何華;趙利;王亞妮;;異基因外周血干細胞移植ABO血型不合輸血的護理[A];第10屆全國實驗血液學會議論文摘要匯編[C];2005年

2 倪建萍;;247例母嬰ABO血型不合的新生兒中血清學檢測結果分析[A];2007年浙江省醫(yī)學檢驗學學術年會論文匯編[C];2007年

3 左江濤;;ABO亞型漏檢原因分析[A];中國輸血協(xié)會第五屆輸血大會論文專集（摘要篇）[C];2010年

4 侯光偉;姜先華;;ABO基因分型及其在法醫(yī)學中的應用[A];第二次全國法醫(yī)物證學學術交流會論文集[C];1998年

5 郎興瑩;尹建平;;ABO血型基因研究進展[A];中國輸血協(xié)會第五屆輸血大會論文專集（摘要篇）[C];2010年

6 章衛(wèi)平;王健民;宋獻民;馮曹波;錢寶華;李紅梅;丁曉勤;;ABO血型不合異基因移植對紅系造血重建的影響[A];第九屆全國實驗血液學會議論文摘要匯編[C];2003年

7 劉福昌;;南昌地區(qū)獻血人群ABO亞型2例分析[A];第五次全國中青年檢驗醫(yī)學學術會議論文匯編[C];2006年

8 喻瓊;蘇宇清;梁延連;孫革;李大成;;ABO血型缺失天然抗體分子機制的研究[A];中國輸血協(xié)會第五屆輸血大會論文專集（摘要篇）[C];2010年

9 劉鐘瀚;王麗紅;李健;;ABO亞型的血型血清學分析[A];中國輸血協(xié)會第三屆輸血大會論文專輯[C];2004年

10 唐綺;李國良;孫愉;肖莉;郭忠慧;;ABO疑難血型鑒定1例[A];中國輸血協(xié)會第三屆輸血大會論文專輯[C];2004年

相關重要報紙文章 前8條

1 張林　記者 劉曉軍;ABO聯(lián)盟：集成關鍵技術 協(xié)同創(chuàng)新發(fā)展[N];科技日報;2009年

2 駐京記者 王丹;ABO的集群經(jīng)驗[N];醫(yī)藥經(jīng)濟報;2007年

3 ;產(chǎn)前診治ABO溶血[N];廣東科技報;2000年

4 季濤;可同時進行STR和ABO基因分型[N];人民公安報;2008年

5 本報記者 項錚;ABO抱團打天下[N];科技日報;2011年

6 本報記者 項錚;ABO啟動“新發(fā)傳染病快速應急反應體系”[N];科技日報;2013年

7 記者 匡玉邋通訊員 陳書云 王鐵石;南華大學成功施行ABO血型不相容腎移植[N];湖南日報;2008年

8 王明;Abo油氣田開發(fā)進入第二階段[N];中國石油報;2004年

相關博士學位論文 前10條

1 翟婭菲;ABO血型抗原的生物法合成及其與腸道菌關系的研究[D];山東大學;2015年

2 王麗;高壓下ABO_4型鎢（鉬）酸鹽的結構和電學性質研究[D];吉林大學;2017年

3 劉長利;北京地區(qū)ABO血型系統(tǒng)分子生物學研究與應用[D];中國人民解放軍軍醫(yī)進修學院;2007年

4 李海英;新型苯并VA嗪衍生物ABO靶向Annexin A7和抑制動脈硬化的機制研究[D];山東大學;2013年

5 劉武;諾如病毒與組織血型抗原結合機制的研究[D];中國科學技術大學;2015年

6 徐昌隆;組織血型抗原基因多態(tài)性與潰瘍性結腸炎的相關性研究[D];武漢大學;2014年

7 韓薇;人血型抗原A糖基轉移酶表達重組質粒體外轉染人乳腺癌細胞MDA-MB-231的抗腫瘤效應研究[D];吉林大學;2017年

8 車輯;去除ABO血型抗體制備通用型血液制劑的研究[D];中國人民解放軍軍醫(yī)進修學院;2008年

9 馮宇;Li~+-Al~(3+)共摻雜ABO_3鐵電陶瓷的電性能與壓電機制[D];哈爾濱工業(yè)大學;2017年

10 劉峰;ABO基因相關多態(tài)性位點在ABO血型不合HSCT移植后嵌合體定量分析中的研究[D];華中科技大學;2011年

相關碩士學位論文 前10條

1 傅倩晰;ABO、ADAMTS7基因遺傳變異與中國漢族人群冠心病易感性的關聯(lián)研究[D];重慶醫(yī)科大學;2015年

2 嚴家煒;ABO血型不合對單份非血緣臍血移植療效的影響[D];安徽醫(yī)科大學;2016年

3 Effat Un Nesa;ABO血型對肺癌患者生存的影響[D];山東大學;2016年

4 劉利祥;ABO、Rh血型相關調(diào)查及建立Rh表型庫在安全輸血中的重要意義[D];天津醫(yī)科大學;2016年

5 王s，

本文編號：1362528