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海豚链球菌(Streptococcus iniae)是引起鱼类链球菌病的主要致病菌,对牙鲆(Paralichthys olivaceus)等养殖鱼类造成巨大危害。本文制备了海豚链球菌强毒株NUF849的福尔马林灭活全菌(FKC)、胞外产物(ECPs)及全菌与胞外产物的混合物(FKC+ECPs),以其免疫牙鲆,在免疫后第42天分别以海豚链球菌NUF849和NUF812攻毒,并在免疫前以及免疫后第7、14、21、28、35、42天与攻毒后第7天取样,分析3种免疫原以及免疫后再攻毒所诱发的血清抗体应答。结果显示,免疫后牙鲆体内产生了分别针对FKC和ECPs的特异性抗体,且各免疫组中FKC诱导的抗体水平较ECPs高,2种抗原间存在一定程度的交叉反应;FKC+ECPs组的两种抗体水平显著高于其他组(P<0.05);攻毒后第7天2种抗体水平都显著升高(P<0.05),并且免疫原来源菌株NUF849攻毒组的2种抗体水平高于非免疫原来源菌株NUF812攻毒组。攻毒后存活牙鲆的血清对NUF849、NUF812全菌蛋白及其胞外产物进行Western-blot分析,结果显示抗血清与全菌蛋白的阳性反应条带位于25~100kDa,与胞外产物的阳性反应条带位于18~100kDa。本文结果表明灭活海豚链球菌与胞外产物都能够诱发牙鲆产生特异性抗体,二者混合物的免疫效果更好,且NUF849来源的免疫原可以刺激牙鲆产生针对NUF812菌株的交叉保护抗体,为牙鲆海豚链球菌疫苗成分的筛选提供了基础资料。
Streptococcus iniae is the main causative agent of streptococcus faecalis and causes great harm to farmed fish such as Paralichthys olivaceus. In this paper, FKC, ECPs and FKC + ECPs of the Streptococcus viridans virulent strain NUF849 were prepared, which were used to immunize flounder, The mice were challenged with Streptococcus pulex putida NUF849 and NUF812 respectively on the 42nd day after immunization, and samples were taken before immunization and on the 7th, 14th, 21st, 28th, 35th and 42nd days after challenge and on the 7th day after the challenge, Serum antibody responses elicited by virulence and challenge. The results showed that specific antibody against FKC and ECPs was produced in flounder after immunization, and the level of antibody induced by FKC in each immunized group was higher than that of ECPs. There was a certain degree of cross-reaction between the two antigens; in FKC + ECPs group The levels of the two antibodies were significantly higher than those of the other groups (P <0.05). The levels of the two antibodies were significantly increased on the 7th day after challenge (P <0.05), and the levels of the two antibodies in the immunized strain NUF849 The non-immunogenic strains NUF812 origin of the challenge group. Western-blot analysis of the full-length NUF849, NUF812 whole-cell proteins and the extracellular products of the surviving flounder serum after challenge showed that the positive reaction bands of the antiserum and the whole-cell protein ranged from 25 to 100 kDa and were positive for the extracellular product The reaction band is located between 18 and 100 kDa. The results of this study indicate that inactivation of S. iniae and extracellular products can induce specific antibody production in flounder, the mixture of the two immune effect is better, and NUF849-derived immunogen can stimulate flounder to produce cross-protection antibodies against NUF812 strains, Provided the basic information for the screening of the components of the flounder, S. iniae.