摘要
[Objectives] This study aimed to analyze the biological activity and immunogenicity of extracellular products from Streptococcus iniae .[Methods] S. iniae was incubated with brain heart infusion agar medium （BHIA ＋ 4% calf serum） for 60 h. The bacterial liquid was rinsed with PBS, centrifuged, and filtered through microporous filtering film to collect extracellular products （ECPs）.[Results] Extracellular proteinase （ECPase） of S. iniae exhibited amylase, protease, lecitinase, gelatinase, lipase activities and hemolytic activity but had no urease activity. EDTA, DTT and PMSF could reduce ECPase activity to 72.4%, 77.6% and 72.4%, respectively. Cu^2＋ , Ca 2＋ , K^＋ and Mg^2＋ exhibited an inhibitory effect on ECPase activity, whereas Fe^3＋ , Co^2＋ and Mn^2＋ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Two-dimensional electrophoresis was performed to analyze the main protein of ECPs. The results indicated that there are 12 main bands of ECPs, and the molecular weights mainly ranged between 28-68 kDa. About 120 protein spots were detected, and the molecular weights mainly ranged between 26-95 kDa. The mouse anti- S. iniae was used for Western-blot analysis of ECPs, and the results showed that there were four proteins, with molecular weights of 26, 37, 95, and 97 kDa, respectively. The pathogenicity assay indicated that ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.[Conclusions] This study provided a certain theoretical basis for revealing the pathogenic mechanism of Streptococcus iniae .
[Objectives] This study aimed to analyze the biological activity and immunogenicity of extracellular products from Streptococcus iniae .[Methods] S. iniae was incubated with brain heart infusion agar medium （BHIA ＋ 4% calf serum） for 60 h. The bacterial liquid was rinsed with PBS, centrifuged, and filtered through microporous filtering film to collect extracellular products （ECPs）.[Results] Extracellular proteinase （ECPase） of S. iniae exhibited amylase, protease, lecitinase, gelatinase, lipase activities and hemolytic activity but had no urease activity. EDTA, DTT and PMSF could reduce ECPase activity to 72.4%, 77.6% and 72.4%, respectively. Cu^2＋ , Ca 2＋ , K^＋ and Mg^2＋ exhibited an inhibitory effect on ECPase activity, whereas Fe^3＋ , Co^2＋ and Mn^2＋ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Two-dimensional electrophoresis was performed to analyze the main protein of ECPs. The results indicated that there are 12 main bands of ECPs, and the molecular weights mainly ranged between 28-68 kDa. About 120 protein spots were detected, and the molecular weights mainly ranged between 26-95 kDa. The mouse anti- S. iniae was used for Western-blot analysis of ECPs, and the results showed that there were four proteins, with molecular weights of 26, 37, 95, and 97 kDa, respectively. The pathogenicity assay indicated that ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.[Conclusions] This study provided a certain theoretical basis for revealing the pathogenic mechanism of Streptococcus iniae .
基金
Supported by Natural Science Foundation of Guangdong Province(2017A030313174)
Natural Science Foundation of Guangdong Ocean University(C17379)
Undergraduate Innovative and Entrepreneurial Team Project(CCTD201802)
Science and Technology Program of Guangdong Province(2015A020209163)
Special Fund for Construction of Fishery Port and Development of Fishery Industry of Guangdong Province(A201708A05)
