鸭疫里默氏菌CH-1株脂多糖合成相关基因lpsA的功能研究

发布时间：2018-04-05 07:24

本文选题：鸭疫里默氏菌　切入点：脂多糖合成基因　出处：《四川农业大学》2015年硕士论文

【摘要】：鸭疫里默氏菌(Riemerella anatipestifer, RA)主要引起鸭、鹅、火鸡以及其他禽类严重的败血症。鸭感染该病后以典型的纤维素性渗出为主要特征,病变主要体现为纤维素性心包炎、肝周炎、气囊炎及脑膜炎等。该病在世界范围内广泛分布,给养禽业带来重大的经济损失。该菌血清型众多,且不同血清型之间不产生交叉保护力,对该病的致病机制研究较少。因此,在生产上,对该病的预防及治疗相当困难。脂多糖(Lipopolysaccharide, LPS),亦称内毒素,是细菌的重要致病因子之一。目前就脂多糖的化学结构,生物合成,遗传调控、致病机制以及免疫等方面受到广泛的关注。对大肠杆菌、沙门氏菌、铜绿假单胞菌等致病菌的脂多糖结构认识较为清楚,可以为病原菌的预防及治疗提供新的途径。目前就鸭疫里默氏菌的脂多糖结构、合成及遗传调控尚未见相关报道。为了研究鸭疫里默氏菌分子致病机制,本实验构建了脂多糖合成修饰基因的缺失株,初步研究了lpsA基因与脂多糖合成及细菌致病性的关系。lpsA基因编码一种脂多糖合成修饰蛋白,功能预测显示该基因在RA脂多糖合成过程中起糖基转移作用,属于糖基转移酶超家族GTB型。本研究主要采用自杀质粒同源重组的基因缺失手段成功构建了血清1型鸭疫里默氏菌的lpsA基因突变株。首先采用PCR分别扩增出RA CH-1的lpsA基因上下游各600bp左右的片段,同时以pYES1 new质粒扩增壮观霉素抗性基因片段,然后利用重叠PCR,将3个片段形成融合片段,再与自杀性质粒pYA4278相连。通过双亲滤膜结合转移法,将构建好的自杀性质粒导入RACH-1株,通过同源重组将抗性片段替换lpsA目的基因构建缺失株。然后通过对缺失株及亲本株的生物学特性进行比较研究,其中包括测定生长曲线、API 20NE及Biolog生化鉴定、药敏实验、遗传稳定性、细胞黏附及侵袭力以及LDso测定等。同时采用多种提取方法提取了亲本株及突变株的脂多糖,通过SDS-PAGE、银染以研究lpsA基因对脂多糖结构的影响。此外,本实验还以热酚水法粗提的RA的脂多糖为免疫原制备了兔抗脂多糖的高免血清。利用制备的抗血清,进行亲本株及缺失株与抗体的凝集试验。实验结果表明lpsA基因缺失后,对RA菌株的生长、毒力等影响不显著,但对菌株的生化特性及耐药性等有一定影响,即缺失株不能发酵或利用某些碳源物质,包括酸化葡萄糖、D-果糖、D-半乳糖、D-海藻糖等。同时对磺胺异恶唑、氟苯尼考、诺氟沙星等抗生素的耐受性减弱。通过对粗提的脂多糖进行SDS-PAGE凝胶电泳、银染,初步研究结果显示,lpsA基因缺失株的脂多糖银染条带与亲本株没有明显差异。以RA亲本株脂多糖制备的多克隆抗体能与lpsA基因缺失株发生凝集反应。结论：lpsA基因缺失后,RA菌株的生长、毒力等不受影响。IpsA基因缺失株脂多糖银染条带与亲本株相似,脂多糖抗原性未发生变化。该基因参与了菌体的糖代谢过程,但是它在RA脂多糖合成及修饰过程中的具体功能还有待深入研究。
[Abstract]:Riemerlla antipestifer (Riemerella anatipestifer RA) is mainly caused by the ducks, geese, turkeys and other poultry. Severe sepsis duck infected with the disease with typical fibrinous lesions as the main feature, mainly reflected the fibrinous pericarditis, perihepatitis, airsacculitis and meningitis. The disease is widely distributed in the world the poultry industry, bring significant economic losses. The bacteria in many serotypes, and cross protection is not generated between different serotypes, of less study on pathogenic mechanism of the disease. Therefore, in practice, it is difficult to prevention and treatment of the disease. Lipopolysaccharide (Lipopolysaccharide, LPS), also called endotoxin, is one of the an important pathogenic factor of bacteria. The chemical structure, lipopolysaccharide biosynthesis, genetic regulation, pathogenic mechanism and immune aspects of attention. In Escherichia coli, Salmonella, Pseudomonas aeruginosa Such as the structure of lipopolysaccharide pathogen clear understanding, can provide a new way for the prevention and treatment of pathogens. The LPS structure of Riemerella anatipestifer, synthesis and genetic regulation has not been reported. In order to study the molecular mechanism of Riemerella anatipestifer, this experiment was constructed to modify gene deletion mutant lipopolysaccharide synthesis a preliminary study on the lpsA gene, and lipopolysaccharide synthesis and pathogenicity of bacteria.LpsA gene encoding a lipopolysaccharide synthesis of modified protein, gene function prediction showed that the glycosyltransferases in RA lipopolysaccharide synthesis process, which belongs to the glycosyltransferase superfamily of type GTB. This research mainly adopts the Dutch act means gene deletion of homologous recombination plasmid the successful construction of the lpsA gene of Riemerella anatipestifer serotypes 1 mutant strains were amplified by PCR. The lpsA gene of RA CH-1 downstream of the 600bp pieces At the same time, with the pYES1 new plasmid amplification spectinomycin resistance gene fragment by overlapping PCR, then, 3 fragments form a fusion fragment, and is connected with the plasmid pYA4278. Dutch act combined transfer method by parent membrane, the constructed plasmid into RACH-1 strain Dutch act, through homologous recombination fragment will replace the lpsA resistance to gene deletion strains. Then through the comparative study on the biological characteristics of mutant and parental strains, including the determination of growth curve, API 20NE and Biolog biochemical identification, drug sensitivity test, genetic stability, cell adhesion and invasion force and determination of LDso. At the same time using a variety of extraction methods of the parental strain and mutant strain lipopolysaccharide, by SDS-PAGE silver staining to study effects of lpsA gene on LPS structure. In addition, this experiment also to lipopolysaccharide hot phenol water extract of RA Rabbit anti lipopolysaccharide preparation of immunogens The hyperimmune serum. Using antiserum agglutination test, strain and parental strain and antibodies were missing. The experimental results show that lpsA gene deletion, the growth of strain RA, the toxicity was not affected, but has a certain effect on the biochemical characteristics of strains and drug resistance, the mutant can not ferment or use some carbon the source material, including acid glucose, D- fructose, D- galactose, D- trehalose and so on. At the same time, sulfamethoxazole, florfenicol, tolerance decreased norfloxacin antibiotics. Through SDS-PAGE gel electrophoresis, silver staining of the crude lipopolysaccharide, preliminary results show that the lpsA gene deletion mutant lipopolysaccharide a silver staining and there is no significant difference in parental strain. The polyclonal antibody was prepared by lipopolysaccharide RA parental can agglutinate with lpsA gene deletion strains. Conclusion: lpsA gene deletion strain RA, the growth of virulence was not affected by.IpsA The silver stained bands of LPS were similar to those of the parental strains, but the antigenicity of LPS was not changed. This gene is involved in the process of glucose metabolism, but its specific function in the process of RA lipopolysaccharide synthesis and modification is still to be further studied.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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