本文選題：鴨疫里默氏桿菌　切入點：煙酰胺酶　出處：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：隨著規(guī)�；B(yǎng)鴨業(yè)的發(fā)展,我國已成為世界上的水禽養(yǎng)殖大國。鴨細菌性傳染病是嚴重危害我國水禽養(yǎng)殖業(yè)發(fā)展的細菌傳染病。鴨疫里默氏桿菌病(又稱為鴨傳染性漿膜炎)是由鴨疫里默氏桿菌(Riemerella anatipestifer,RA)引起的鴨、鵝等多種禽類的一種急性或慢性接觸性傳染,是危害水禽業(yè)發(fā)展最嚴重的細菌傳染病之一。目前已報道鴨疫里默氏桿菌的血清型有21個,各血清型間的交叉保護力較低導(dǎo)致疫苗在預(yù)防鴨疫里默氏桿菌感染的應(yīng)用受到較大限制。至今,鴨疫里默氏桿菌致病機理仍不清楚。Purser,J.E等人已經(jīng)證明煙酰胺酶(Nicotinamidase,NAMase)是導(dǎo)致萊姆病細菌感染的一個重要的酶。在流產(chǎn)布魯氏菌中,NAMase為流產(chǎn)布氏桿菌復(fù)制所必需。人類紅細胞感染瘧原蟲過程中,體內(nèi)煙酰胺活性和NAD+合成量顯著提高。盡管煙酰胺酶在不同生物中具有重要作用,但其精確的催化機制尚未完全闡明。研究鴨疫里默氏桿菌煙酰胺酶的生物學(xué)和酶學(xué)特性將有助于控制該病。本研究對鴨疫里默氏桿菌Yb2株的煙酰胺酶進行了克隆表達以及生物學(xué)和酶學(xué)特性分析。研究結(jié)果為鴨疫里默氏桿菌免疫保護蛋白和分子致病機理的研究奠定了基礎(chǔ)。本研究分四部分進行:1.NAMase生物信息學(xué)分析生物信息學(xué)研究表明,NAMase由201個氨基酸組成,分子式為C1014H1560N262O307S9,在溶液中較穩(wěn)定,蛋白總體親水性較高。重組蛋白為非分泌型蛋白,無信號肽,亞細胞定位于細胞質(zhì)中;無跨膜區(qū);蛋白B細胞表位分析:13-15,20-27,50-67,86,107-121,177-187,氨基酸位置可能為B細胞線性表位;蛋白質(zhì)二級結(jié)構(gòu)包含5個螺旋,7個折疊,12個無規(guī)則卷曲;蛋白三級結(jié)構(gòu)為文章圖所示。2.鴨疫里默氏桿菌NAMase基因的克隆及在E.coli中的表達以血清型2型鴨疫里默氏桿菌Yb2菌株基因組為模板,進行PCR擴增得到606 bp大小的NAMase基因,連接到原核表達載體pET28a,構(gòu)建重組質(zhì)粒pET28a-NAMase,然后轉(zhuǎn)化至大腸桿菌BL21(DE3),采用酶切和序列測定鑒定,獲得正確結(jié)果后,加入IPTG誘導(dǎo)表達蛋白。本研究成功表達大小約為22.1 KDa的重組蛋白His-NAMase,該重組蛋白以可溶性方式表達。用Ni+親和層析柱純化后獲得高純度重組蛋白His-NAMase,為酶學(xué)及免疫學(xué)特性研究奠定了基礎(chǔ)。3.鴨疫里默氏桿菌NAMase多克隆抗體制備與NAMase亞細胞定位分析純化的NAMase與佐劑乳化后皮下免疫接種新西蘭大白兔,制備多克隆抗體,用間接ELISA法測定抗體效價,Western-blot檢測多克隆抗體的免疫原性。實驗結(jié)果表明,經(jīng)純化的NAMase免疫后,新西蘭大白兔可產(chǎn)生特異性抗體,抗體效價達到1:32000。經(jīng) SDS-PAGE、Western blot 檢測 NAMase 蛋白位于細胞質(zhì)中。4.鴨疫里默氏桿菌NAMase酶學(xué)性質(zhì)分析經(jīng)測定NAMase最適pH值為6;最適溫度為40℃;該酶的活性受金屬離子Zn~(2+)、Cu~(2+)、Fe~(3+)的影響。
[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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