ABO血型抗原的生物法合成及其与肠道菌关系的研究

发布时间：2018-01-01 02:23

本文关键词：ABO血型抗原的生物法合成及其与肠道菌关系的研究　出处：《山东大学》2015年博士论文　论文类型：学位论文

【摘要】：糖类,广泛存在于自然界的所有生物体中,不仅是不可或缺的生物体组成成分,也是生物体维持生命活动的主要能量来源。此外,糖类往往作为信息分子与其它分子相结合来发挥其重要的生物学功能。因此,以糖类为研究对象的糖生物学是了解生命过程和发展人类健康事业的重要科学,并日益被科学界和生物医药公司所重视。ABO血型系统是人类认识最早且最为普遍的血型分类系统,可分为A、B、O和AB型四种,其血型抗原的本质是糖蛋白和糖脂,血型是由糖链末端的组成和结构决定的。组成ABO血型的单糖包括GlcNAc、Gal、GalNAc及fucose。目前,血型与疾病的关系已成为研究的热点,现已知胃癌、胰腺癌、冠心病、高血压、渗出性中耳炎和病毒性脑炎等疾病均在不同血型的人群中发病率不同,但相关研究仍处在病例统计分析水平,两者之间相互作用的具体机理仍然不清楚。ABO血型抗原对于血型与人体健康的研究至关重要,因此,ABO血型糖抗原及其它生物活性糖类的制备对于其在生物医学中的研究和应用显得尤为重要。由于酶法合成寡糖具有高效化学和立体构型选择性且经济省时,此法已趋向于替代传统的化学合成方法。但由于Leloir型糖基转移酶的底物糖核苷酸的数量有限且价格昂贵,限制了此法在大量合成生物活性寡糖方面的应用,严重阻碍了糖及糖缀合物在医药领域的研究和应用。糖核苷酸是单糖的活化形式,是由核苷一磷酸或核苷二磷酸与单糖的异头碳连接形成的。糖核苷酸主要作为糖基转移酶的底物发挥作用,不仅可以作为糖基供体来研究糖基转移酶的生化性质,还可以通过对糖核苷酸中的单糖进行修饰从而形成糖基转移酶抑制剂或将单糖衍生物添加到糖缀合物中来标记糖链或研究糖类的代谢途径。生物酶法和微生物发酵法都有其各自的优势,均是有潜力实现糖核苷酸工业化生产的方法。肠道菌群与宿主之间是互利共生的,是人体不可分割的一个重要组成部分,相当于人体的一个重要“功能器官”,在宿主的代谢、免疫调控、防御、大脑发育以及人类行为等多方面都发挥着不可替代的作用。肠道菌群紊乱也与多种疾病的发生发展密切相关。在肠道菌群与宿主共同进化的过程中,来自宿主的遗传因素、生活环境、饮食以及社会行为等内在或外在因素都会影响肠道菌群的组成和结构。肠道菌表面多糖有脂多糖、荚膜多糖和胞外多糖,其中很多糖结构由于与人类ABO血型抗原相似而具有血型物质活性,能与ABO天然抗体发生免疫反应。因此探究ABO血型对肠道菌群组成的影响,不仅可以解释血型与疾病易感性之间的关联,又可为ABO天然抗体起源的假说提供一个有力的佐证。本论文的目标之一是简化UDP-GlcNAc的酶法合成并通过小规模制备得到其纯品。UDP-GlcNAc是GlcNAc转移酶催化的糖基化反应发生的先决条件。本课题组之前已用来自长双歧杆菌的GlcNAc1位激酶(NahK)和来自大肠杆菌的截短的核苷酸焦磷酸化酶(truncated GlmU)合成了 UDP-GlcNAc,这里我们将这两种酶进行了融合表达并摸索了融合酶的最适反应条件为pH 8.0,温度40℃。之后,以GlcNAc, ATP和UTP为底物,用融合酶进行一步法反应生成了UDP-GlcNAc,产率为88%。反应液经过碱性磷酸酶消化降解副产物后再经过分子筛分离纯化。最终总产率可达77%,得到的产品的纯度可达90%以上。含有fucose的生物分子拥有多种生物学功能,而能够催化其合成的糖基转移酶的必要糖基供体底物为GDP-Fuc。在本论文的第三章中,我们将来自于脆弱拟杆菌的合成GDP-Fuc的补救途径引入了大肠杆菌中。此外,由于GTP是GDP-Fuc生物合成的一个重要底物,我们过表达了 GTP生物合成补救途径中的一些酶来增强它的合成。对加强表达的酶进行不同的组合,形成了不同的工程菌株,并通过摇瓶发酵比较了它们生成产物的能力。其中同时表达Fkp, Gpt, Gmk和Ndk的菌株产量最高,每克干重细菌内含有4.6±0.22μmol,是只表达Fkp菌株的4.2倍。通过分批补料发酵,GDP-Fuc的产量进一步提高到6.6±0.14 μmol/g。与利用从头合成途径发酵生产GDP-Fuc相比,此方法不仅产量更高而且可以用来合成在fucose残基上有化学修饰的GDP-Fuc类似物。影响肠道菌群组成的因素多种多样,某些细菌的表面多糖具有ABO血型抗原活性,那么血型对肠道菌群组成的影响引起了我们的关注。本论文的第四章分析了提取于不同血型的171个个体的肠道菌群16SrDNA序列。不同血型人群的肠道菌群组成基本相似,但在不同的分类水平上存在一些差别。拟杆菌门、互养菌门以及柔膜菌门在不同血型人群中有差别,而其它门均无显著性差异。在各分组间α多样性在A型血人群的肠道菌群中最低,此外我们还发现A型和AB型人群的肠道菌中有益菌的数量比其它血型少。为了进一步探索肠道菌群适应宿主选择压的机制,我们进一步检测了不同组的肠道菌群的血型抗原活性,发现A型血人群的肠道菌群有更高的A抗原活性,B型血人群的肠道菌群有更高的B抗原活性,而O型和AB型血人群的肠道菌群拥有相同的A和B抗原活性。这些结果说明ABO血型的确能够影响肠道菌群的组成,细菌表面多糖的血型抗原活性不同是造成这种现象的原因之一,这就为血型与疾病易感性的机制研究指明了一个新的方向。总之,本学位论文利用酶法和微生物发酵法合成了两种糖核苷酸。通过构建融合酶简化了 UDP-GlcNAc的酶法合成,对大肠杆菌进行了代谢工程改造,获得了高效合成GDP-Fuc的工程菌株。确定了 ABO血型对肠道菌群组成的影响并从细菌表面多糖与血型抗原的关系入手,初步解释了血型与肠道菌群间的相互关系。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R457.11;R37

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