创伤弧菌FJ03-X2荚膜多糖的提取和免疫原性分析

发布时间：2018-10-23 14:24

【摘要】： 创伤弧菌(Vibrio vulnificus)是严重危害水产动物的人兽致病菌。荚膜多糖(Capsular polysaccharide,CPS)是创伤弧菌重要的毒力因子和抗原成分,在创伤弧菌的致病过程中起重要作用,为了了解创伤弧菌CPS的免疫原性,本研究以鳗源创伤弧菌强毒株FJ03-X2为研究对象,提取纯化了创伤弧菌CPS,并对其成分、分子量,毒力和免疫原性进行了分析。紫外吸收分光光度法测定CPS样品,其主要峰值在200nm,基本符合多糖吸收峰的特征;SDS-PAGE的双氨银染图谱未见明显的蛋白质和脂多糖条带;琼脂糖凝胶电泳结果显示CPS样品含少量核酸成分。结果表明提取得到较纯的CPS。通过SDS-PAGE和阿尔新蓝-双氨银染法分析CPS,结果表明,CPS的相对分子量分布范围为311～20 KDa;CPS可分为高分子量成分和低分子量成分;高分子量成分仅用阿尔新蓝染色即可在凝胶上清晰显示,呈现为连续的弥散状区带,其含量较高,相对分子量范围为311～43 KDa;低分子量成分则需阿尔新蓝-双氨银染法染色才能在凝胶上显示,其含量较低,相对分子量范围为43～20 KDa。用CPS经腹腔注射免疫昆明鼠,通过菌体凝集反应、ELISA和Dot ELISA等方法分析CPS对鼠的免疫原性,结果显示,鼠抗CPS血清可和一定浓度的创伤弧菌菌株FJ03-X2菌液发生凝集反应,凝集效价为2~(4～5);ELISA结果表明鼠抗CPS血清的抗体效价可达1∶12 800。初步说明,鳗源创伤弧菌菌株FJ03-X2的CPS对小鼠具有免疫原性,能够诱导小鼠产生特异性体液免疫应答。毒力试验表明,CPS对欧鳗无直接致病力。CPS经腹腔注射免疫接种欧鳗,Dot ELISA结果显示,CPS不能激发试验欧鳗产生体液免疫应答;免疫保护试验表明,CPS不能诱导试验欧鳗产生保护性免疫应答。
[Abstract]:Vibrio vulnificus (Vibrio vulnificus) is a human animal pathogen that seriously endangers aquatic animals. Capsule polysaccharide (Capsular polysaccharide,CPS) is an important virulence factor and antigenic component of Vibrio vulnificus, which plays an important role in the pathogenesis of Vibrio vulnificus. In order to understand the immunogenicity of Vibrio vulnificus CPS, a virulent strain FJ03-X2 from Vibrio vulgaris was studied in this study. CPS, was extracted and purified from Vibrio vulnificus and its components, molecular weight, virulence and immunogenicity were analyzed. UV absorption spectrophotometry showed that the main peak value of CPS was 200 nm, which basically accorded with the characteristics of polysaccharide absorption peak, and no obvious protein and lipopolysaccharide bands were found in the silver staining spectrum of SDS-PAGE. Agarose gel electrophoresis showed that CPS samples contained a small amount of nucleic acids. The results showed that pure CPS. was obtained by extraction. The results of CPS, analysis by SDS-PAGE and Alxinole-bisamino-silver staining showed that the relative molecular weight distribution range of CPS was 31120 KDa;CPS, which could be divided into high molecular weight component and low molecular weight component. The high molecular weight components can be clearly displayed on the gel by Alxin blue staining, showing a continuous dispersion zone, and the content of the high molecular weight components is relatively high. The relative molecular weight of the low molecular weight components in the range of 311C 43 KDa; required Albumin blue-diamino silver staining to show a lower content on the gel, with a relative molecular weight range of 43 ~ 20 KDa.. Kunming mice were immunized by intraperitoneal injection of CPS. The immunogenicity of CPS to mice was analyzed by cell agglutination reaction, ELISA and Dot ELISA. The results showed that the anti-CPS serum of mice could agglutinate with FJ03-X2 of a certain concentration of Vibrio vulnificus strain. The agglutination titer was 2 ~ (4); ELISA. The results showed that the antibody titer of mouse anti CPS serum could reach 1:12 800. The preliminary results showed that the CPS of Vibrio vulnificus strain FJ03-X2 had immunogenicity to mice and could induce specific humoral immune response in mice. The virulence test showed that CPS had no direct pathogenicity to eel. CPS was inoculated intraperitoneally with eel, Dot ELISA. The results showed that CPS could not stimulate humoral immune response of eel. Immune protection test showed that CPS could not induce protective immune response in eel.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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