两株致病性哈维氏弧菌胞外产物的特性分析
CHARACTERISTICS OF THE EXTRACELLAR PRODUCTS OF TWO PATHOGENIC VIBRIO HARVEYI STRAINS
-
摘要: 采用硫酸铵分级盐析从哈维氏弧菌EcGY020401株和SpGY020601株培养物中提取其胞外产物,并对胞外产物的一些特性进行了分析。结果表明,两株哈维氏弧菌的胞外产物均具有较强的毒力,对鲫鱼的半致死剂量LD50为7.1g蛋白/g鱼体重;均具有淀粉酶、酪蛋白酶、脂肪酶活性,而无脲酶和明胶酶活性;除EcGY020401株胞外产物对斜带石斑(Epinephelus coioides)红细胞有较强溶血作用外,对小鼠、鲫、人O型血等红细胞的溶血性都较弱或无;对草鱼吻端成纤维细胞(PSF)都具有细胞毒性。此外,经聚丙烯酰胺葡聚糖凝胶柱层析和离子交换柱层析,对Sp-GY020601株的胞外产物中蛋白酶进行了初步提纯,SDS-PAGE分析得一分子量约为38Kda的蛋白酶,该蛋白酶对鲫鱼有致死毒性,半致死剂量为3.3g蛋白/g鱼体重。Abstract: Vibrio harveyi is a kind of important pathogenic bacteria for seawater animals, such as prawn, crab, clam, and ormer. Along with the development of seawater fish cage-culture in China, Vibrio harveyi also becomes a main cause of disease of cage-culture fish, including Lutianus erythopterus, Seriola dumerili, Lateoabrax japonicus, Epinephlus fario and Epinephelus coioides. Some researches indicate that Vibrio harveyi can secrete extracellular products and result in the damnification of host tissue or result in the death of host when seriously. Two Vibrio harveyi strains, EcGY020401 and SpGY020601, which were isolated from diseased Epinephelus coioides and Lutianus erythopterus in south China, were identified after morphological observation, biochemical characteristic analysis, and 16srRNA or HSP60 gene sequence detection. Both strains can cause death to freshwater fish and marine fish, such as crucian carp ( Carassius auratus) and estuary cod( Epinephelus coioides) through artificial injection. In order to understand their patho-genetic mechanism, extracellular products(ECPs) of both strains were extracted with ammonium sulfate precipitation, and some characteristics of their ECPs such as exoenzymatic, hemolytic, toxic and cytotoxic activities were analyzed. Virulence experiments showed that ECPs of the two strains had quite strong virulence to crucian carp in that their LD50 to crucian carp was about 7.1μg-protein/ g fish weight . Both strains’ECPs have the activity of amylase, protease and lipase, but have not the activity of gelatinase and urease. Except that the ECP of strain EcGY020401 has stronger haemolytic activity to the red cell of grouper, the ECPs have very weak hemolysis to erythrocytes of human, mouse, estuary cod and crucian carp. Cytotoxic activity to the fish cell line PSF was detected in both ECPs, and the lowest cytotoxic concentration to PSF is 2.0μg/ mL and 4.0μg/ mL, respectively. A main protease in ECPs produced by strain SpGY020601 was also purified following the steps of ammonium sulfate precipitation, gel filtration and anion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE) showed the estimated molecular mass of the protease is about 38KDa. The protease is lethal to crucian carp and the LD50 is 3.3μg-protein/ g. Phenylmethanesulfonyl fluoride (PMSF) and Ethylene diamine tetraacetic acid (EDTA) had a little inhibitory effects on the activity of the protease, while Ca2+ 、Zn2+ 、Mg2+ can enhance the protease’activity. These results indicated that the protease might be an alkaline cysteine protease.
-
Keywords:
- Vibrio harveyi /
- Extracellular products /
- Hemolysis /
- Cytotoxicity /
- Pathogenicity /
- Protease
-
-
[1] Liu X Z, Zou XL, Mo X Y, et al. Study on the pathogen of vibriosis isolated from diseased Epinephelus sp. in cage mariculture[J ].Tropic Oceanoogy, 1994, 13(1): 81-86[刘秀珍,邹晓理,莫小燕,等. 海水网箱养殖石斑鱼病原菌研究. 热带海洋,1994,13(1): 81-86]
[2] Wu H B, Pan J P. Studies on the pathogenic bacteria of the vibriosisof Seriola dumerili in marine cage culture[J ]. Journal of Fisheriesof China, 1997, 21(2): 171-174[吴后波,潘金培. 海水网箱养殖高体 弧菌病致病菌研究. 水产学报, 1997, 21( 2):171-174]
[3] Xiao H, Li J, Wang X H, et al. Studies on pathogens of rotted gilland rotted caudal fins of seaperch( Lateolabrax japonicus) fry[J ].Journal of Ocean University of Qingdao, 1999, 29 (1): 87-93[肖慧,李军,王祥红,等. 鲈鱼苗烂鳃、烂尾病病原菌的研究. 青岛海洋大学学报(自然科学版),1999, 29(1): 87-93]
[4] Chang J B, Gong X H, Sun F X, et al . Preliminary studies on apathogen of vibriosis of cultured Paralichthys olivaceus [J]. MarineFisheries Research, 2001, 22 (1): 37-42[常建波,宫向红,孙逢贤,等. 养殖牙鲆弧菌病病原菌初步研究. 海洋水产研究,2001, 22 (1): 37-42]
[5] Xu B F, Lin N F, Yang J X, et al. Isolation, identification andpathogenicity analysis of Vibrio parahaemolyticus from Pseudosciaenacrocea[J]. Fujian Journal of Agriculture Sciences, 2002, 17 (3):174-177[许斌福,林能锋,杨金先,等. 大黄鱼副溶血弧菌的分离、鉴定及致病力分析. 福建农业学报, 2002, 17(3):174-177]
[6] Bradshaw J G, Shaj D B, Wehby AJ, et al. Thermal inactivation ofthe Kanagawa hemolysin of Vibrio parahaemolyticus in buffer andshrimp[J]. Journal of Food Scinece, 1984, 49: 184-187
[7] Shinichi M, Kazuhiro S, et al. Purification and Characterization of aHemolysin Produced by Vibro mimicu [J]. Infection and immunity,1997, 65(5): 1830-1835
[8] Lee K K, Yu S R, Liu P C. Alkaline serine protease is an exotoxinof Vibrio alginolyticus in kuruma prawn, Penaeus japonicus[J].Curr Microbiol, 1997, 34: 110-117]
[9] Mou HJ, Li J, Bao ZM, et al. Pathogenicity of extracellular products of Vibrio parahaemolyticus to Penaeus chinensis[J ]. Oceanolo2gia Et Limnologia Sinica, 2000, 31 (3): 273-279 [牟海津,李筠,包振民,等. 副溶血弧菌胞外产物对中国对虾的致病性分析. 海洋与湖沼, 2000, 31(3): 273-279]
[10] Wu H B, Pan J P. Virulence mechanisms of pathogenic vibrio [J] .Acta Hydrobiologica Sinica, 2003, 27 (4): 422-426 [吴后波,潘金培. 病原弧菌的致病机理. 水生生物学报,2003, 27 (4):422-426 ]
[11] Wu H B, Pan J P. The characteristics of the exotoxin Vm-Pm pro2duced by Vibrio mimicus [J]. Acta Hydrobiologica Sinica, 2004, 28(4): 409-412[吴后波,潘金培. 海水养殖真鲷弧菌病病原菌外毒素的理化特性. 水生生物学报,2004,28(4):409-412]
[12] Chen X G, Wu S Q, Shi C B, et al. Isolation and identification ofpathogenetic Vibrio harveyi from estuary cod, Epinephelus coioides[J]. Journal of Fishery Sciences of Chin, 2004, 11 (4): 313-317[陈献稿,吴淑勤,石存斌,等. 斜带石斑鱼病原菌(哈维氏弧菌) 的分离与鉴定. 中国水产科学, 2004, 11 (4): 313-317]
[13] Wei Y X, Wang J C, Cheng DL, et al. Purification and property of extracellular protease from Vibrio anguillarum [J]. Chin J Appl Env2iron, 2002, 8 (4): 414-418[魏玉西,汪靖超,程殿林,等. 鳗弧菌胞外产物中蛋白酶的纯化及其性质. 应用与环境生物学报,2002, 8(4): 414-418]
[14] Lee K K, Chen YL, Liu P C. Hemostasis of Tiger Prawn Penaeusmonodon affected by Vibrio harveyi, extracellular products and a toxiccysteine protease [J]. Blood Cells Molecules and Diseases, 1999, 25(13): 180-192
[15] Mo ZL, Chen S Y, Zhang PJ. Properties of proteolytic toxin of Vibrio anguillarum from diseased flounder[J]. Chinese Journal ofOceanology and Limnology, 2002, 20(4): 316-322
[16] Liu P C, Lee K K, Tu C C, et al. Purification and characterizationof a cysteine protease produced by pathogenic luminous Vibrio harveyi[J]. Curr. Microbiol, 1997, 35: 32-39
计量
- 文章访问数: 952
- HTML全文浏览量: 4
- PDF下载量: 738
下载: