三种弧菌共同抗原的筛选和鉴定
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摘要
近年来,随着水产养殖业的迅猛发展,养殖规模不断扩大,养殖密度也逐渐增加,导致养殖水域环境逐年恶化,疾病发生越来越频繁,给养殖业造成了巨大的经济损失。而细菌性病原是引起海水鱼暴发性疾病的主要病原,弧菌则是一种主要的病原菌,这其中又以哈氏弧菌、溶藻弧菌、副溶血弧菌最为严重。
本文对哈氏弧菌、溶藻弧菌、副溶血弧菌全菌免疫反应性蛋白进行了研究,鉴定出了5个三种弧菌共同免疫反应性蛋白,并对其中一种蛋白质的免疫保护作用进行了验证。
研究发现:
哈氏弧菌免疫反应性蛋白:提取哈氏弧菌的全菌蛋白,通过双向电泳、Western Blot得到哈氏弧菌全菌蛋白2-DE图谱和哈氏弧菌全菌蛋白免疫转印图谱,用ImageMaster 2D Platinum图像分析软件匹配得到45个蛋白质点,进行质谱鉴定后得到40个蛋白,这其中有4对蛋白虽在胶上的位置不同,但鉴定为同一种蛋白,有一个蛋白点没有在数据库中查找到相对应的蛋白质,另外,蛋白编号H17和H19是同一蛋白的两个亚基。
溶藻弧菌免疫反应性蛋白:提取溶藻弧菌的全菌蛋白,通过双向电泳、Western Blot得到溶藻弧菌全菌蛋白2-DE图谱和溶藻弧菌全菌蛋白免疫转印图谱,用ImageMaster 2D Platinum图像分析软件匹配得到40个蛋白质点,进行质谱鉴定后得到36种蛋白,这其中有2对蛋白虽在胶上的位置不同,但鉴定为同一种蛋白;有一个蛋白点没有鉴定出来;有一个蛋白点没有在数据库中查找到相对应的蛋白质;另外,蛋白编号A24和A29是同一蛋白的两个亚基
副溶血弧菌免疫反应性蛋白:提取副溶血弧菌的全菌蛋白,通过双向电泳、Western Blot得到副溶血弧菌全菌蛋白2-DE图谱和副溶血弧菌全菌蛋白免疫转印图谱,用ImageMaster 2D Platinum图像分析软件匹配得到58个蛋白质点,从中挑选了17个蛋白质点进行质谱鉴定,得到14种蛋白,这其中有2对蛋白虽在胶上的位置不同,但鉴定为同一种蛋白;有一个蛋白点没有在数据库中查找到相对应的蛋白质。三种弧菌共同抗原:用ImageMaster 2D Platinum图像分析软件对三种弧菌全菌蛋白2-DE图谱上标记了数字的蛋白点进行配对分析,匹配得到10个蛋白质点;分别对三种弧菌的这10个蛋白点进行质谱鉴定,最终鉴定得到5种蛋白质是三种弧菌共同的免疫反应性蛋白质,分别是dihydrolipoamide dehydrogenase、succinate dehydrogenase flavoprotein subunit、elongation factor Ts、glyceraldehyde-3-phosphate dehydrogenase、triosephosphate isomerase。
免疫保护率:从胶面提取蛋白dihydrolipoamide dehydrogenase腹腔注射斜带石斑鱼,4周后分别用哈氏弧菌、溶藻弧菌、副溶血弧菌攻毒,计算免疫保护率。结果表明:由蛋白dihydrolipoamide dehydrogenase制备的疫苗对这三种弧菌的免疫保护率分别达到70%、60%、70%。
In recent years, with the rapid development of aquaculture, diseases of farmed fish occur more frequently, which has caused tremendous economic losses for aquaculture industry. Bacterial diseases caused by Vibrio species are known to be the main reason for the mortality and Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus are proved to be the most important pathogens of marine fish in South China according to the formerly research on the epidemiology of Vibrio diseases.
In this paper, the immuno-reaction proteins of V. harveyi, V. alginolyticus and V. parahaemolyticus recoginzed by rabbit anti-V. harveyi antibody have been identified by immunoproteomics method. Furthermore the common antigens of these three Vibrio species have been screened and the protective function of one of the common antigens has been studied.
Immuno-reaction Proteins of V. harveyi: whole cell extract protein of V. harveyi through two-dimensional electrophoresis, Western Blot to get 2-DE of whole cell protein of V. harveyi map and 2-DE Western blotting of whole cell protein of V. harveyi map. Through Image Master 2D Platinum to match to get 45 protein sopts; through mass spectrometry identification we get 40 proteins. Although four pairs of protein spots in the gel get different locations, they are identified as the same protein; one protein spot can not be found the corresponding protein in the database; in addition, H17 and H19 are two subunits of the same protein.
Immuno-reaction Proteins of V. alginolyticus: whole cell extract protein of V. alginolyticus through two-dimensional electrophoresis, Western Blot has got 2-DE of whole cell protein of V. alginolyticus map and 2-DE Western blotting of whole cell protein of V. alginolyticus map. Through Image Master 2D Platinum to match we get 40 sopts, through mass spectrometry identification we get 36 proteins. Although two pairs of protein spots in the gel have different locations, they are identified as the same protein; one protein spot can not be found the corresponding protein in the database; a protein spots has not been identified out; in addition, A24 and A29 are two subunits of the same protein.
Immuno-reaction Proteins of V. parahaemolyticus: whole cell extract protein of V. parahaemolyticus through two-dimensional electrophoresis, Western Blot has got 2-DE of whole cell protein of V. parahaemolyticus map and 2-DE Western blotting of whole cell protein of V. parahaemolyticus map. Through Image Master 2D Platinum to match we get 58 sopts, Checking 17 spots out of them through mass spectrometry identification, we get 14 kinds of proteins. Although two pairs of protein spots in the gel have get the different locations, they are identified as the same protein; one protein spot can not be found the corresponding protein in the database.
Common antigens of the three Vibrio species.: By ImageMaster 2D Platinum analyzes of three Vibrio sp. whole cell protein 2-DE maps, we found 10 protein spots. Each of the three Vibrio sp. of these 10 protein spots were identified separately by MS, we got five kinds of proteins. They are common antigens of the three Vibrio sp.: dihydrolipoamide dehydrogenase, succinate dehydrogenase flavoprotein subunit, elongation factor Ts, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase.
Relative percentage survival (RPS): Firstly, Dihydrolipoamide dehydrogenase was injected intraperitoneally into each E. coioides Cuvier; then three Vibrio sp. was injected intraperitoneally into each E. coioides Cuvier after 4 weeks. Cumulative mortality was recorded over two weeks. The different RPSs were 70% (V. harveyi), 60% (V. alginolyticus), and 70% (V. parahaemolyticus).
本文对哈氏弧菌、溶藻弧菌、副溶血弧菌全菌免疫反应性蛋白进行了研究,鉴定出了5个三种弧菌共同免疫反应性蛋白,并对其中一种蛋白质的免疫保护作用进行了验证。
研究发现:
哈氏弧菌免疫反应性蛋白:提取哈氏弧菌的全菌蛋白,通过双向电泳、Western Blot得到哈氏弧菌全菌蛋白2-DE图谱和哈氏弧菌全菌蛋白免疫转印图谱,用ImageMaster 2D Platinum图像分析软件匹配得到45个蛋白质点,进行质谱鉴定后得到40个蛋白,这其中有4对蛋白虽在胶上的位置不同,但鉴定为同一种蛋白,有一个蛋白点没有在数据库中查找到相对应的蛋白质,另外,蛋白编号H17和H19是同一蛋白的两个亚基。
溶藻弧菌免疫反应性蛋白:提取溶藻弧菌的全菌蛋白,通过双向电泳、Western Blot得到溶藻弧菌全菌蛋白2-DE图谱和溶藻弧菌全菌蛋白免疫转印图谱,用ImageMaster 2D Platinum图像分析软件匹配得到40个蛋白质点,进行质谱鉴定后得到36种蛋白,这其中有2对蛋白虽在胶上的位置不同,但鉴定为同一种蛋白;有一个蛋白点没有鉴定出来;有一个蛋白点没有在数据库中查找到相对应的蛋白质;另外,蛋白编号A24和A29是同一蛋白的两个亚基
副溶血弧菌免疫反应性蛋白:提取副溶血弧菌的全菌蛋白,通过双向电泳、Western Blot得到副溶血弧菌全菌蛋白2-DE图谱和副溶血弧菌全菌蛋白免疫转印图谱,用ImageMaster 2D Platinum图像分析软件匹配得到58个蛋白质点,从中挑选了17个蛋白质点进行质谱鉴定,得到14种蛋白,这其中有2对蛋白虽在胶上的位置不同,但鉴定为同一种蛋白;有一个蛋白点没有在数据库中查找到相对应的蛋白质。三种弧菌共同抗原:用ImageMaster 2D Platinum图像分析软件对三种弧菌全菌蛋白2-DE图谱上标记了数字的蛋白点进行配对分析,匹配得到10个蛋白质点;分别对三种弧菌的这10个蛋白点进行质谱鉴定,最终鉴定得到5种蛋白质是三种弧菌共同的免疫反应性蛋白质,分别是dihydrolipoamide dehydrogenase、succinate dehydrogenase flavoprotein subunit、elongation factor Ts、glyceraldehyde-3-phosphate dehydrogenase、triosephosphate isomerase。
免疫保护率:从胶面提取蛋白dihydrolipoamide dehydrogenase腹腔注射斜带石斑鱼,4周后分别用哈氏弧菌、溶藻弧菌、副溶血弧菌攻毒,计算免疫保护率。结果表明:由蛋白dihydrolipoamide dehydrogenase制备的疫苗对这三种弧菌的免疫保护率分别达到70%、60%、70%。
In recent years, with the rapid development of aquaculture, diseases of farmed fish occur more frequently, which has caused tremendous economic losses for aquaculture industry. Bacterial diseases caused by Vibrio species are known to be the main reason for the mortality and Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus are proved to be the most important pathogens of marine fish in South China according to the formerly research on the epidemiology of Vibrio diseases.
In this paper, the immuno-reaction proteins of V. harveyi, V. alginolyticus and V. parahaemolyticus recoginzed by rabbit anti-V. harveyi antibody have been identified by immunoproteomics method. Furthermore the common antigens of these three Vibrio species have been screened and the protective function of one of the common antigens has been studied.
Immuno-reaction Proteins of V. harveyi: whole cell extract protein of V. harveyi through two-dimensional electrophoresis, Western Blot to get 2-DE of whole cell protein of V. harveyi map and 2-DE Western blotting of whole cell protein of V. harveyi map. Through Image Master 2D Platinum to match to get 45 protein sopts; through mass spectrometry identification we get 40 proteins. Although four pairs of protein spots in the gel get different locations, they are identified as the same protein; one protein spot can not be found the corresponding protein in the database; in addition, H17 and H19 are two subunits of the same protein.
Immuno-reaction Proteins of V. alginolyticus: whole cell extract protein of V. alginolyticus through two-dimensional electrophoresis, Western Blot has got 2-DE of whole cell protein of V. alginolyticus map and 2-DE Western blotting of whole cell protein of V. alginolyticus map. Through Image Master 2D Platinum to match we get 40 sopts, through mass spectrometry identification we get 36 proteins. Although two pairs of protein spots in the gel have different locations, they are identified as the same protein; one protein spot can not be found the corresponding protein in the database; a protein spots has not been identified out; in addition, A24 and A29 are two subunits of the same protein.
Immuno-reaction Proteins of V. parahaemolyticus: whole cell extract protein of V. parahaemolyticus through two-dimensional electrophoresis, Western Blot has got 2-DE of whole cell protein of V. parahaemolyticus map and 2-DE Western blotting of whole cell protein of V. parahaemolyticus map. Through Image Master 2D Platinum to match we get 58 sopts, Checking 17 spots out of them through mass spectrometry identification, we get 14 kinds of proteins. Although two pairs of protein spots in the gel have get the different locations, they are identified as the same protein; one protein spot can not be found the corresponding protein in the database.
Common antigens of the three Vibrio species.: By ImageMaster 2D Platinum analyzes of three Vibrio sp. whole cell protein 2-DE maps, we found 10 protein spots. Each of the three Vibrio sp. of these 10 protein spots were identified separately by MS, we got five kinds of proteins. They are common antigens of the three Vibrio sp.: dihydrolipoamide dehydrogenase, succinate dehydrogenase flavoprotein subunit, elongation factor Ts, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase.
Relative percentage survival (RPS): Firstly, Dihydrolipoamide dehydrogenase was injected intraperitoneally into each E. coioides Cuvier; then three Vibrio sp. was injected intraperitoneally into each E. coioides Cuvier after 4 weeks. Cumulative mortality was recorded over two weeks. The different RPSs were 70% (V. harveyi), 60% (V. alginolyticus), and 70% (V. parahaemolyticus).
引文
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