嗜水气单胞菌主要外膜蛋白对欧洲鳗鲡的免疫保护试验
MAJOR OUTER MEMBRANE PROTEIN(MOMP)OF AEROMONAS HYDROPHILA INDUCED PROTECTIVE IMMUNITY TO EUROPEAN EELS( ANGUILLIA ANGU ILLIA)
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摘要: 制备出4株分属3个不同血清型嗜水气单胞菌的主要外膜蛋白(MOMP)免疫刺激复合物(ISCOMs)亚单位疫苗,腹腔注射免疫欧洲鳗鲡,ELISA法测得免疫后第14d和第30d所采集的血清的平均抗体效价在1∶320-1∶1280之间。免疫印迹结果显示,在印迹膜上对应的位置均可检测到MOMP的主要蛋白条带。第40d的免疫保护试验表明,免疫欧鳗对TPS-30菌株约100倍半数致死剂量的攻击无免疫保护作用,其中的3组被测免疫欧鳗对TPS-30菌株约10倍半数致死剂量的攻击的免疫保护率皆在80%以上。上述试验结果提示,嗜水气单胞菌的主要外膜蛋白是重要的保护性抗原,在制备成ISCOMs亚单位疫苗的情况下,较小的剂量免疫一次即可显示出较高的相对免疫保护率,且没有明显的血清型特异性。Abstract: This study was performed to estimate the hypothesis that the major outer membrane(MOMP) of Aeromonas hydrophila is possibly the important common protective antigen to fish1MOMP of four strains Aeromonashydrophila which belong to three different serogroups were prepared by treatment with 1. 2% N-lauroylsarcosinate detergent, and then concentrated and purified by utracentrifuging1Structural analysis exhibited that threeof them havethe sameMOMP pattern and themajor protein bands were 40 kilodalton and 43-kilodalton in SDS-PAGE gel1Strain WC-2 has another MOMP pattern To enhance the immunogenicity of MOMP,MOMP-ISCOMs(Immune-stimulating complex, ISCOMs)were prepared by dialysis assay Transmission electron micrograph showed that MOMP-ISCOMs prepared in this study had some basicmatrix profiles contrast to the typical ISCOMs European eels(Anguillia anguillia)were immunized by intraperitoneal injection(i1p)with a dose of 20Lg per fish with these MOMP-ISCOMs1Serum were collected on fourteenth day and thirtieth day, respectively.The average serum antibody titers ranged from 1B320) 1B1280 by enzyme linked immunosorbent assay (ELISA)1Immunoblot indicated that all these four MOMP and four MOMP-ISCOMs could be completely recognized by the corresponding anti serum Challenge experiment was carried out on fortieth day with the virulent strain TPS-30 whose 50% ethal dosage(LD50) to eels(20-30g per fish) is about 3.0 106CFU(clony forming unit) per fish. No protective effect were observed when challenged with a dose of 3.0 108CFU/fish, but over 80% relative percent survival(RPS)were observed when given a challenge with a dose of 3.0 107CFU/fish.All these results showed that MOMP of A1hydrophila used as ISCOMs form is important protective antigen. A low immunized dosage could stimulate eels to produce high protective immunity, and no distinct difference was observed among different O-serogroups strains