海水鱼类主要病原弧菌致病性及外膜蛋白研究
摘要
海水养殖向高密度、集约化发展的同时，鱼类病害问题日益突出。其中弧菌病害是危害海水养殖鱼类最严重的细菌病害之一，许多弧菌可感染多种鱼类并造成大量死亡，本文主要对山东地区海水养殖场分离的致病弧菌进行了以下几方面的研究：
从自然发病大菱鲆体内分离到病原菌TL01，经生理生化鉴定和分子生物学鉴定为鳗弧菌；对从自然发病的鲈鱼、大菱鲆体内分离的病原鳗弧菌W-1、TL01及哈维氏弧菌SF-1的致病性研究表明，鳗弧菌W-1、TL01对牙鲆、大菱鲆有较强的致病性。W-1对牙鲆和大菱鲆腹腔感染的半数致死量LD_(50)分别是2.06×10~4cfu／g和3.04×10~3cfu／g；TL01对牙鲆和大菱鲆腹腔感染的半数致死量LD_(50)分别是5.09×10~4cfu／g和2.32×10~1cfu／g；哈维氏弧菌SF-1对牙鲆和大菱鲆腹腔感染的半数致死量LD_(50)分别是9.65×10~3cfu／g和8.63×10~2cfu／g。
进行了鳗弧菌TL01、W-1抗血清制备，血清学实验表明，鳗弧菌W-1和TL01都属于O2血清型，哈维氏弧菌SF-1与鳗弧菌各血清型菌株之间无明显交叉反应。
对13株弧菌属的不同种的菌株、10个不同血清型的鳗弧菌标准菌株、9株哈维氏弧菌和4株副溶血弧菌的标准菌株的外膜蛋白分别提取并进行了分析；结果表明13株弧菌属的不同种的菌株外膜蛋白的SDS-PAGE图谱各不相同。不同菌种间主要的外膜蛋白有较大差异，一般有4-5条，分子量范围为25kD～70kD。
10种血清型的鳗弧菌的主要外膜蛋白的图谱各不相同。每个血清型的鳗弧菌标准菌株外膜蛋白有2-6条，分子量主要在25kD-70kD之间，但所有鳗弧菌均有1～2条主要外膜蛋白，分子量界于40kD和55kD之间，且在大部分菌株中其中一条的分子量在40kD左右。从当地分离的病原菌W-1和TL01最主要的外膜蛋白为40kD左右，另外还各有一条主要外膜蛋白带，分子量分别为53kD和44kD左右。
哈维氏弧菌菌株之间主要外膜蛋白的分子量相差比较大，没发现有各菌株间共同的主要蛋白，主要外膜蛋白有3-6条，分子量集中在30-48kD的区域。副溶血弧菌几株菌的外膜蛋白图谱总体比较相似，条带数比较多，一般多于3-5条，主
中国梅洋大学硕士研究生论文
要外膜蛋白分子量界于30kD一okD。
研究了不同培养基和培养条件对弧菌对外膜蛋白表达的影响，结果表明在缺
铁基础培养基CMg中37℃条件下鳗弧菌W一1、TL01表达了一条分子量较小的主要
外膜蛋白，分子量为35kD左右。
用SePhadex一G100、头phaeryl 5100凝胶层析和DEAE.SePharose Fast Flow、
DEAE一纤维素层析对鳗弧菌W一1和TL01的主要外膜蛋白进行了分离，均未取得
理想的分离效果。最后采用SDS-PAGE电泳分离后，直接在凝胶上切下目的蛋白，
得到了电泳纯的鳗弧菌主要外膜蛋白，其分子量为35kD。
用鳗弧菌外膜蛋白以注射接种的方式对海水养殖大菱虾鱼苗进行免疫，4周后
检测血清中特异性抗体，然后用鳗弧菌悬液(3 .7xl了cfu/ml)0.2ml腹腔注射感
染免疫鱼，结果表明用鳗弧菌外膜蛋白免疫后能刺激大菱虾产生特异性免疫反应，
免疫4周人工感染后的免疫保护力为45.45%。
Culture of marine fish progresses to the level of high density and intensity, accompanied by a prominence of disease problem. Some strains of Vibrio species are the most serious pathogens, which can infect a wide range of fish species and cause mass death. The present study is placed upon the following aspects:
Two pathogenic Vibrio anguillarum strains W-1, TL01 were originally isolated from diseased seabass (Lateolabrax japonicus) and diseased turbot (Scophthalmus maximus) in Qingdao of Shandong province, China. Pathogenicity of V.anguillarum W-l, TL01, and a pathogenic Vibrio harveyi SF-1 to turbot ((Scophthalmus maximus) and flunder (Paralichthys olivaceus) are studied by intraperitoneal injection. Vibrio anguillarum W-l and TL01 are highly pathogenic to turbot and flunder. LDso of V.anguillarum W-l to flunder and turbot are 2.06 104cfu/g and3.04 103cfu/g fish respectively; LD50 of TL01 were5.09 104cfu/g and 2.32 101cfu/g fish respectively; LD50 of Vibrio harveryi to flunder and turbot are 9.65 103cfu/g 8.63 102cfu/g fish respectively.
Serological analysis show that antisera of W-l and TL01 both react with serotype O2 type strain VIB2, but not with other serotype type strains, W-l and TL01 have good cross-reaction with each other, which suggest that they both belong to serotype O2 strain.
Outer membrane proteins (OMP) of 13 Vibrio type strains, 12 strains of Vibrio anguillarum, 9 Vibrio harveyi and 4 Vibrio parahaemolyticus bacteria are extracted and analyzed by SDS-PAGE.
The OMP profiles of 13 Vibrio type strains are heterogeneous, four to five
main OMPs were found, with the molecular weight ranged between 25kD and 70kD.
OMP profiles of 10 serotype strains of V. anguillarum are different from each other, each strain had two to six dominant proteins of 25-70kD. One to two common protein was found among the 10 V. anguillarum strains, and of them one is 40kD in most strains. W-l and TL01 have different OMP profiles from each other, but both of them had the 40kD-55kD common proteins.
The main OMPs profiles of Vibrio harveyi strains are different from each other, Each strain had three to six main proteins, and the molecular weights range from 30kD to 48kD, No common OMP is found among these strains.
Vibrio parahaemolyticus have the similar OMP patterns. Each strain have three to five proteins. The molecular weights ranged from 30kD to 50kD.
A 35kD protein is produced by W-land TL01 when cultured in CM9 at 37 癈. Gel filtration with Sephadex-G10(K Sephacryl S-10CK DEAE-Sepharose Fast Flow N DEAE-cellulose are used to purify the main OMP of V.anguillarum W-l but no method is efficient. The induced 35kD protein of W-l is separated by SDS-PAGE.
Turbot fries were vaccinated with partly purified OMPs of V.anguillarum W-l via intraperitoneal injection. A dosage of 0.2 ml of V.anguiUarum W-l (3.7 l08cfu/ml) was intraperitoneally injected to the vaccinated fish and control group 4 weeks later. The results show that specific immune reaction can be stimulated by the proteins. Specific antibody could be detected after injection immunization and the relative percentage survival (RPS) was 45.45%.
从自然发病大菱鲆体内分离到病原菌TL01，经生理生化鉴定和分子生物学鉴定为鳗弧菌；对从自然发病的鲈鱼、大菱鲆体内分离的病原鳗弧菌W-1、TL01及哈维氏弧菌SF-1的致病性研究表明，鳗弧菌W-1、TL01对牙鲆、大菱鲆有较强的致病性。W-1对牙鲆和大菱鲆腹腔感染的半数致死量LD_(50)分别是2.06×10~4cfu／g和3.04×10~3cfu／g；TL01对牙鲆和大菱鲆腹腔感染的半数致死量LD_(50)分别是5.09×10~4cfu／g和2.32×10~1cfu／g；哈维氏弧菌SF-1对牙鲆和大菱鲆腹腔感染的半数致死量LD_(50)分别是9.65×10~3cfu／g和8.63×10~2cfu／g。
进行了鳗弧菌TL01、W-1抗血清制备，血清学实验表明，鳗弧菌W-1和TL01都属于O2血清型，哈维氏弧菌SF-1与鳗弧菌各血清型菌株之间无明显交叉反应。
对13株弧菌属的不同种的菌株、10个不同血清型的鳗弧菌标准菌株、9株哈维氏弧菌和4株副溶血弧菌的标准菌株的外膜蛋白分别提取并进行了分析；结果表明13株弧菌属的不同种的菌株外膜蛋白的SDS-PAGE图谱各不相同。不同菌种间主要的外膜蛋白有较大差异，一般有4-5条，分子量范围为25kD～70kD。
10种血清型的鳗弧菌的主要外膜蛋白的图谱各不相同。每个血清型的鳗弧菌标准菌株外膜蛋白有2-6条，分子量主要在25kD-70kD之间，但所有鳗弧菌均有1～2条主要外膜蛋白，分子量界于40kD和55kD之间，且在大部分菌株中其中一条的分子量在40kD左右。从当地分离的病原菌W-1和TL01最主要的外膜蛋白为40kD左右，另外还各有一条主要外膜蛋白带，分子量分别为53kD和44kD左右。
哈维氏弧菌菌株之间主要外膜蛋白的分子量相差比较大，没发现有各菌株间共同的主要蛋白，主要外膜蛋白有3-6条，分子量集中在30-48kD的区域。副溶血弧菌几株菌的外膜蛋白图谱总体比较相似，条带数比较多，一般多于3-5条，主
中国梅洋大学硕士研究生论文
要外膜蛋白分子量界于30kD一okD。
研究了不同培养基和培养条件对弧菌对外膜蛋白表达的影响，结果表明在缺
铁基础培养基CMg中37℃条件下鳗弧菌W一1、TL01表达了一条分子量较小的主要
外膜蛋白，分子量为35kD左右。
用SePhadex一G100、头phaeryl 5100凝胶层析和DEAE.SePharose Fast Flow、
DEAE一纤维素层析对鳗弧菌W一1和TL01的主要外膜蛋白进行了分离，均未取得
理想的分离效果。最后采用SDS-PAGE电泳分离后，直接在凝胶上切下目的蛋白，
得到了电泳纯的鳗弧菌主要外膜蛋白，其分子量为35kD。
用鳗弧菌外膜蛋白以注射接种的方式对海水养殖大菱虾鱼苗进行免疫，4周后
检测血清中特异性抗体，然后用鳗弧菌悬液(3 .7xl了cfu/ml)0.2ml腹腔注射感
染免疫鱼，结果表明用鳗弧菌外膜蛋白免疫后能刺激大菱虾产生特异性免疫反应，
免疫4周人工感染后的免疫保护力为45.45%。
Culture of marine fish progresses to the level of high density and intensity, accompanied by a prominence of disease problem. Some strains of Vibrio species are the most serious pathogens, which can infect a wide range of fish species and cause mass death. The present study is placed upon the following aspects:
Two pathogenic Vibrio anguillarum strains W-1, TL01 were originally isolated from diseased seabass (Lateolabrax japonicus) and diseased turbot (Scophthalmus maximus) in Qingdao of Shandong province, China. Pathogenicity of V.anguillarum W-l, TL01, and a pathogenic Vibrio harveyi SF-1 to turbot ((Scophthalmus maximus) and flunder (Paralichthys olivaceus) are studied by intraperitoneal injection. Vibrio anguillarum W-l and TL01 are highly pathogenic to turbot and flunder. LDso of V.anguillarum W-l to flunder and turbot are 2.06 104cfu/g and3.04 103cfu/g fish respectively; LD50 of TL01 were5.09 104cfu/g and 2.32 101cfu/g fish respectively; LD50 of Vibrio harveryi to flunder and turbot are 9.65 103cfu/g 8.63 102cfu/g fish respectively.
Serological analysis show that antisera of W-l and TL01 both react with serotype O2 type strain VIB2, but not with other serotype type strains, W-l and TL01 have good cross-reaction with each other, which suggest that they both belong to serotype O2 strain.
Outer membrane proteins (OMP) of 13 Vibrio type strains, 12 strains of Vibrio anguillarum, 9 Vibrio harveyi and 4 Vibrio parahaemolyticus bacteria are extracted and analyzed by SDS-PAGE.
The OMP profiles of 13 Vibrio type strains are heterogeneous, four to five
main OMPs were found, with the molecular weight ranged between 25kD and 70kD.
OMP profiles of 10 serotype strains of V. anguillarum are different from each other, each strain had two to six dominant proteins of 25-70kD. One to two common protein was found among the 10 V. anguillarum strains, and of them one is 40kD in most strains. W-l and TL01 have different OMP profiles from each other, but both of them had the 40kD-55kD common proteins.
The main OMPs profiles of Vibrio harveyi strains are different from each other, Each strain had three to six main proteins, and the molecular weights range from 30kD to 48kD, No common OMP is found among these strains.
Vibrio parahaemolyticus have the similar OMP patterns. Each strain have three to five proteins. The molecular weights ranged from 30kD to 50kD.
A 35kD protein is produced by W-land TL01 when cultured in CM9 at 37 癈. Gel filtration with Sephadex-G10(K Sephacryl S-10CK DEAE-Sepharose Fast Flow N DEAE-cellulose are used to purify the main OMP of V.anguillarum W-l but no method is efficient. The induced 35kD protein of W-l is separated by SDS-PAGE.
Turbot fries were vaccinated with partly purified OMPs of V.anguillarum W-l via intraperitoneal injection. A dosage of 0.2 ml of V.anguiUarum W-l (3.7 l08cfu/ml) was intraperitoneally injected to the vaccinated fish and control group 4 weeks later. The results show that specific immune reaction can be stimulated by the proteins. Specific antibody could be detected after injection immunization and the relative percentage survival (RPS) was 45.45%.
引文
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