海豚链球菌及其胞外产物诱导的牙鲆血清抗体应答分析

Immune Response of Flounder(Paralichthys Olivaceus) to Three Types of Streptococcus Iniae Immunogen

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 iniaeis the major etiological agent of streptococcal disease,which is responsible for hemorrhagic septicemia in fish,particularly olive flounders（Paralichthys olivaceus）.In this paper,healthy flounders were vaccinated with 3types of Streptococcus iniaeimmunogen,which were formalinkilled cells（FKC）,extracellular products（ECPs）and mixture of the former two components（FKC＋ECPs）.The antibody kinetics were detected in the blood sample collected from the caudal veins of flounders before vaccination and on 7d,14 d,21d,28 d,35d,42dpost-vaccination and 7dpost-challenge by the homologous（NUF849）and heterologous isolates（NUF812）respectively.The results showed cross-reaction phenomena were observed between the specific antibodies against FKC and ECPs which were elicited in flounder vaccinated with either FKC or ECPs,and the group immunized with FKC presented a higher antibody titre than ECPs.The antibody levels were significantly higher in the group immunized with FKC＋ECPs than the other two groups（P〈0.05）;Also the levels of antibody to FKC and ECPs were higher in the flounder challenged with NUF849 than NUF812.Furthermore,Western-blot analysis was performed using the anti-sera collected from post-challenge survival flounders reacting with whole cell lysates and ECPs from both NUF849 and NUF812.The result of Western-blot showed that bands of whole cell lysates and ECPs proteins reacting with the sera located from 25 kDa to100kDa and 18 kDa to 100 kDa in the gel respectively.These results showed that specific antibodies could be induced in flounder by either FKC or ECPs,but the mixtures of the two immunogens could produce higher titre antibody;Additionally,cross-protective antibodies against NUF812 could be elicited in flounders vaccinated with immunogens from NUF849.This study will give insights into the selection of immunogens as effective vaccine components to against Streptococcus iniae.