温度对溶藻弧菌HY9901株致病性相关蛋白的调控
REGULATION OF PATHOGENICITY ASSOCIATED PROTEIN BY TEMPERATURE IN VIBRIO ALGINOLYTICU SHY9901 STRAIN
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摘要: 将溶藻弧菌HY9901株置于15℃、20℃、28℃、37℃四个温度下培养,18h时分别测定细菌含量,胞外蛋白酶(Extracelluar protease,ECPase)表达量以及胞外产物(Extracellular product,ECP)对动物的致死性。结果表明,随着温度升高,菌液浓度、ECPase的活性以及ECP对小鼠和红笛鲷的致死率先升高后下降。28℃培养的细菌浓度、ECPase的活性以及ECP对小鼠和红笛鲷的致死率均为最大。用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析不同温度培养下细菌的ECP和外膜蛋白(Outer membrane protein,OMP),结果显示,不同温度培养的溶藻弧菌ECP和OMP电泳图谱不同。溶藻弧菌主要ECP的分子量为70kD和62kD。ECP 70kD在20℃时的表达量最大,而28℃时ECP62 kD的蛋白浓度最高。45kD OMP随着温度升高,其合成量增大,28℃时达最大值;而40kD和34kD OMP则随着温度的升高而减弱。通过研究电泳纯化的ECP 62kD和ECP 70kD的酶活性和致死试验,证明ECP 62kD具有很强的酶活性和毒性,而ECP 70kD仅具有弱的酶活性和毒性。Abstract: Vibrio alginolyticus is one of the most important opportunist pathogen,which exists extensively in seawater,and causes diarrhea in human and vibriosis in maricultured animals.The pathogenesis of V.alginolyticus disease is not clearly understood.In this article,the expression of some factors by V.alginolyticus HY9901 strain cultured in 15℃,20℃,28℃ and 37℃ was analyzed,respectively.The effects of culture temperature on bacterial growth were studied on TSA media.Poteolytic activity of Extracelluar protease(ECPase)were detected by the method of Azocasein.Virulence of extracelluar product(ECP)were studied by using Lethality for mice and Lutjanus sanguineus Cuvier.The results indicated that bacterial growth,poteolytic activity of Extracelluar protease(ECPase) and virulence of ECP all enhanced firstly and then dropped as the temperature rising,and 28℃ was the optimum temperature.ECP and outer membrane protein(OMP) expression were studied by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).It was suggested that the bacteria showed diverse ECP and OMP profiles under different cultural temperatures.The major bands of ECP for V.alginolyticus was 70kD when the cells grown at 20℃,while 62kD ECP at 28℃.45kD OMP became denser when the cells grown temperature was rising,while 40kD and 34kD OMP became slight.The result also showed that SDS-PAGE purified ECP 62kD had strong poteolytic activity and Lethality for animals,and SDS-PAGE purified ECP 70kD had weak poteolytic activity and Lethality for animals.These results suggested that a virulent factor of V.alginolyticus may be induced or enhanced under 28℃.The temperature-regulated factors reported in this paper may be important in the pathogenesis of V.alginolyticus disease.The presented evidence also suggested that V.alginolyticus and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs.
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