摘要
从患腹水症牙鲆脾脏组织中分离得到一株优势菌株CD86,经人工感染实验证实菌株CD86对牙鲆有较高的致病力,其半致死浓度为1.8×105 CFU/尾。通过生理生化特征测定和16SrDNA基因序列分析,判定菌株CD86为鱼肠道弧菌(Vibrio ichthyoenteri)。以福尔马林灭活的菌株CD86全菌分别免疫健康的小鼠和牙鲆,ELISA测定鼠抗血清和牙鲆抗血清效价分别为12 800和1 200。利用间接免疫荧光技术检测2种抗血清与菌株CD86的结合活性,结果显示,2种血清均可同菌体发生阳性反应,其中鼠抗血清阳性反应信号明显强于鱼抗血清。同时,利用Western blotting分析了菌株CD86全菌蛋白的抗原性,结果显示分子量为27、30和37kDa的菌体蛋白条带与2种血清均发生阳性反应;分子量为19、25、28、29和35kDa的菌体蛋白条带仅与鼠抗血清发生阳性反应;分子量为45kDa的菌体蛋白条带只与鱼抗血清发生阳性反应。研究结果表明,牙鲆对菌体抗原产生的抗体应答与小鼠存在明显差异,该发现对研筛鱼用高效的鱼肠道弧菌亚单位疫苗具有重要的参考价值。
A dominant bacterial strain named CD86 was isolated from the spleen of flounder Paralichthys olivaceus with ascites.The artificial infection experiment revealed that strain CD86 was quite virulent to healthy flounder with an LD50 value of 1.8×10^5 CFU/fish.The biochemical test was performed with Rapid ID32 Estrip.The results showed that all the detected indexes of strain CD86 were in agreement with the standard strain.Meanwhile,the sequence of 16 SrDNA of strain CD86 showed the highest similarity with that of Vibrio ichthyoenteri retrieved from the gene bank database.Vibrio phylogenetic tree based on 16 SrDNA sequences also showed that strain CD86 and V.ichthyoenteri clustered into one blade.Combining with its biochemical result we concluded that strain CD86 was actually V.ichthyoenteri.Furthermore,healthy mice and flounder were immunized with formalin inactivated strain CD86.The results of ELISA showed that the titers of the antisera from mice and flounder were determined to be 12 800 and 1 200,respectively.Indirect Immunofluorescence assay showed that antisera from mice and flounder could both react with strain CD86,whereas the mice antiserum showed a stronger reaction compared with flounder antiserum.Meanwhile,Western blotting was employed to analyze the antigenicity of the whole bacterial protein of strain CD86 using the antisera from mice and flounder,and the result showed that these two antisera could both reacted with three proteins with molecular weight（MW）of 27,30 and 37kDa,and the proteins with MW of 19,25,28,29 and 35kDa was only recognized by mice antisera,whereas a 45 kDa protein was only recognized by flounder antisera.These results demonstrated that the antibody response patterns of mice and flounder to bacterial antigen were much different,which has important reference value for screening and developing subunit vaccine of V.ichthyoenteri.
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2016年第10期24-31,共8页
Periodical of Ocean University of China
基金
国家自然科学基金项目(31302216
31472295
31672685)
国家重点基础研究发展规划项目(2012CB114406)
泰山学者特聘专家项目
国家科技支撑计划项目(2012BAD17B01)资助~~
