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

  更多相關(guān)文章： ABO血型 酶法合成 發(fā)酵法合成 細(xì)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(jié)合來發(fā)揮其重要的生物學(xué)功能。因此,以糖類為研究對象的糖生物學(xué)是了解生命過程和發(fā)展人類健康事業(yè)的重要科學(xué),并日益被科學(xué)界和生物醫(yī)藥公司所重視。ABO血型系統(tǒng)是人類認(rèn)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(zhì)是糖蛋白和糖脂,血型是由糖鏈末端的組成和結(jié)構(gòu)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(guān)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(guān)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(jī)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(guān)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(xué)中的研究和應(yīng)用顯得尤為重要。由于酶法合成寡糖具有高效化學(xué)和立體構(gòu)型選擇性且經(jīng)濟(jì)省時,此法已趨向于替代傳統(tǒng)的化學(xué)合成方法。但由于Leloir型糖基轉(zhuǎn)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(yīng)用,嚴(yán)重阻礙了糖及糖綴合物在醫(yī)藥領(lǐng)域的研究和應(yīng)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(zhuǎn)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(zhuǎn)移酶的生化性質(zhì),還可以通過對糖核苷酸中的單糖進(jìn)行修飾從而形成糖基轉(zhuǎn)移酶抑制劑或?qū)翁茄苌锾砑拥教蔷Y合物中來標(biāo)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(dāng)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(guān)。在腸道菌群與宿主共同進(jìn)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(jié)構(gòu)。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(jié)構(gòu)由于與人類ABO血型抗原相似而具有血型物質(zhì)活性,能與ABO天然抗體發(fā)生免疫反應(yīng)。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(guān)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(biāo)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(zhuǎn)移酶催化的糖基化反應(yīng)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(jìn)行了融合表達(dá)并摸索了融合酶的最適反應(yīng)條件為pH 8.0,溫度40℃。之后,以GlcNAc, ATP和UTP為底物,用融合酶進(jìn)行一步法反應(yīng)生成了UDP-GlcNAc,產(chǎn)率為88%。反應(yīng)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(dá)77%,得到的產(chǎn)品的純度可達(dá)90%以上。含有fucose的生物分子擁有多種生物學(xué)功能,而能夠催化其合成的糖基轉(zhuǎn)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(bǔ)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(dá)了 GTP生物合成補(bǔ)救途徑中的一些酶來增強(qiáng)它的合成。對加強(qiáng)表達(dá)的酶進(jìn)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(dá)Fkp, Gpt, Gmk和Ndk的菌株產(chǎn)量最高,每克干重細(xì)菌內(nèi)含有4.6±0.22μmol,是只表達(dá)Fkp菌株的4.2倍。通過分批補(bǔ)料發(fā)酵,GDP-Fuc的產(chǎn)量進(jìn)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(xué)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(xì)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(guān)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(jìn)一步探索腸道菌群適應(yīng)宿主選擇壓的機(jī)制,我們進(jìn)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(jié)果說明ABO血型的確能夠影響腸道菌群的組成,細(xì)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(jī)制研究指明了一個新的方向�？傊�,本學(xué)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(gòu)建融合酶簡化了 UDP-GlcNAc的酶法合成,對大腸桿菌進(jìn)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(xì)菌表面多糖與血型抗原的關(guān)系入手,初步解釋了血型與腸道菌群間的相互關(guān)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R457.11;R37

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3 劉長利;北京地區(qū)ABO血型系統(tǒng)分子生物學(xué)研究與應(yīng)用[D];中國人民解放軍軍醫(yī)進(jìn)修學(xué)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(jī)制研究[D];山東大學(xué);2013年

5 劉武;諾如病毒與組織血型抗原結(jié)合機(jī)制的研究[D];中國科學(xué)技術(shù)大學(xué);2015年

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

7 韓薇;人血型抗原A糖基轉(zhuǎn)移酶表達(dá)重組質(zhì)粒體外轉(zhuǎn)染人乳腺癌細(xì)胞MDA-MB-231的抗腫瘤效應(yīng)研究[D];吉林大學(xué);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(jìn)修學(xué)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(jī)制[D];哈爾濱工業(yè)大學(xué);2017年

10 劉峰;ABO基因相關(guān)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(xué);2011年

相關(guān)碩士學(xué)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(guān)聯(lián)研究[D];重慶醫(yī)科大學(xué);2015年

2 嚴(yán)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(xué);2016年

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

4 劉利祥;ABO、Rh血型相關(guān)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(xué);2016年

5 王s，

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