鸭疫里默氏杆菌烟酰胺酶的原核表达及酶学活性的研究

发布时间：2018-03-17 11:49

  本文选题：鸭疫里默氏杆菌　切入点：烟酰胺酶　出处：《南京农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着规模化养鸭业的发展,我国已成为世界上的水禽养殖大国。鸭细菌性传染病是严重危害我国水禽养殖业发展的细菌传染病。鸭疫里默氏杆菌病(又称为鸭传染性浆膜炎)是由鸭疫里默氏杆菌(Riemerella anatipestifer,RA)引起的鸭、鹅等多种禽类的一种急性或慢性接触性传染,是危害水禽业发展最严重的细菌传染病之一。目前已报道鸭疫里默氏杆菌的血清型有21个,各血清型间的交叉保护力较低导致疫苗在预防鸭疫里默氏杆菌感染的应用受到较大限制。至今,鸭疫里默氏杆菌致病机理仍不清楚。Purser,J.E等人已经证明烟酰胺酶(Nicotinamidase,NAMase)是导致莱姆病细菌感染的一个重要的酶。在流产布鲁氏菌中,NAMase为流产布氏杆菌复制所必需。人类红细胞感染疟原虫过程中,体内烟酰胺活性和NAD+合成量显著提高。尽管烟酰胺酶在不同生物中具有重要作用,但其精确的催化机制尚未完全阐明。研究鸭疫里默氏杆菌烟酰胺酶的生物学和酶学特性将有助于控制该病。本研究对鸭疫里默氏杆菌Yb2株的烟酰胺酶进行了克隆表达以及生物学和酶学特性分析。研究结果为鸭疫里默氏杆菌免疫保护蛋白和分子致病机理的研究奠定了基础。本研究分四部分进行:1.NAMase生物信息学分析生物信息学研究表明,NAMase由201个氨基酸组成,分子式为C1014H1560N262O307S9,在溶液中较稳定,蛋白总体亲水性较高。重组蛋白为非分泌型蛋白,无信号肽,亚细胞定位于细胞质中;无跨膜区;蛋白B细胞表位分析:13-15,20-27,50-67,86,107-121,177-187,氨基酸位置可能为B细胞线性表位;蛋白质二级结构包含5个螺旋,7个折叠,12个无规则卷曲;蛋白三级结构为文章图所示。2.鸭疫里默氏杆菌NAMase基因的克隆及在E.coli中的表达以血清型2型鸭疫里默氏杆菌Yb2菌株基因组为模板,进行PCR扩增得到606 bp大小的NAMase基因,连接到原核表达载体pET28a,构建重组质粒pET28a-NAMase,然后转化至大肠杆菌BL21(DE3),采用酶切和序列测定鉴定,获得正确结果后,加入IPTG诱导表达蛋白。本研究成功表达大小约为22.1 KDa的重组蛋白His-NAMase,该重组蛋白以可溶性方式表达。用Ni+亲和层析柱纯化后获得高纯度重组蛋白His-NAMase,为酶学及免疫学特性研究奠定了基础。3.鸭疫里默氏杆菌NAMase多克隆抗体制备与NAMase亚细胞定位分析纯化的NAMase与佐剂乳化后皮下免疫接种新西兰大白兔,制备多克隆抗体,用间接ELISA法测定抗体效价,Western-blot检测多克隆抗体的免疫原性。实验结果表明,经纯化的NAMase免疫后,新西兰大白兔可产生特异性抗体,抗体效价达到1:32000。经 SDS-PAGE、Western blot 检测 NAMase 蛋白位于细胞质中。4.鸭疫里默氏杆菌NAMase酶学性质分析经测定NAMase最适pH值为6;最适温度为40℃;该酶的活性受金属离子Zn~(2+)、Cu~(2+)、Fe~(3+)的影响。
[Abstract]:With the development of large-scale duck farming, Our country has become a big country of water fowl breeding in the world. Bacterial infectious disease of duck is a bacterial infectious disease that seriously endangers the development of water fowl breeding in our country. Riemer's disease of duck epidemic (also called infectious serositis of duck) is caused by disease of duck Riemer. The duck caused by Riemerella anatipestifera), An acute or chronic contact infection of a variety of birds, including geese, is one of the most serious bacterial infections that harm the development of the waterfowl industry. At present, 21 serotypes of Riemer's disease have been reported. The low cross-protection among serotypes has limited the application of the vaccine in the prevention of Riemer's disease infection. The pathogenic mechanism of Riemer's disease remains unclear. Purserine J.E et al. Has shown that Nicotinamidase NAMaseis an important enzyme that causes bacterial infection of Lyme disease. In brucella abortus, NAMase is necessary for brucellosis replication. Cell infection with Plasmodium, The activity of nicotinamide and the amount of NAD synthesis were significantly increased in vivo, although nicotinase plays an important role in different organisms. However, its precise catalytic mechanism has not been fully elucidated. The study of biological and enzymatic properties of nicotinase from Riemer's disease Duck will be helpful to control the disease. The cloning of nicotinase from Yb2 strain of Riemer Duck was carried out in this study. The results laid a foundation for the study of immune protection protein and molecular pathogenicity of Riemer's disease. This study was divided into four parts: 1. NAMase bioinformatics analysis of bioinformatics. Studies have shown that NAMase consists of 201 amino acids, The molecular formula is C1014H1560N262O307S9, which is stable in solution and has high overall hydrophilicity. Protein B cell epitope analysis showed that the amino acid position of protein B cell epitope may be B cell linear epitope, the protein secondary structure contains 5 helix, 7 folds and 12 irregular curls. The tertiary structure of protein was shown in the paper. 2. Cloning and expression of NAMase gene of Riemer's disease strain in E. coli. The NAMase gene of 606 BP was obtained by PCR amplification using the genome of Yb2 strain of serotype 2 as template. The recombinant plasmid pET28a-NAMasewas constructed by ligation to prokaryotic expression vector pET28a-NAMase. the recombinant plasmid pET28a-NAMasewas then transformed into Escherichia coli BL21DDE3, which was identified by restriction endonuclease digestion and sequencing. The recombinant protein His-NAMasewas successfully expressed with the size of 22.1 KDa. The recombinant protein was expressed in a soluble manner. The recombinant protein His-NAMasewas purified by Ni affinity chromatography. The recombinant protein His-NAMasewas enzymatic and immunological. The preparation of NAMase polyclonal antibody and the subcellular localization of NAMase purified NAMase and adjuvant were subcutaneously immunized with New Zealand white rabbits. Polyclonal antibody was prepared and immunogenicity of polyclonal antibody was detected by indirect ELISA assay and Western-blot. The results showed that New Zealand white rabbit could produce specific antibody after immunizing with purified NAMase. The antibody titer was 1: 32000.The NAMase protein was detected by SDS-PAGEG Western blot in the cytoplasm. The analysis of the enzymatic properties of NAMase of Riemer Ductus was carried out. The optimum pH value of NAMase was 6 and the optimum temperature was 40 鈩，

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