大嘴鲈鱼对奴卡氏菌Nocardiaseriolae之免疫反应及其疫苗之研发.DOCVIP

大嘴鱸魚對奴卡氏菌Nocardia seriolae之免疫反應及其疫苗之研發 Immune response of largemouth bass to Nocardia seriolae and its vaccine development 研究生：黃韻芬 Huang, Yun-Fen 指導教授：陳石柱 Chen, Shih-Chu 【摘要】 本研究引用前人在脈衝式電泳 ( pulse field gel electrophoresis ) 及 API ZYM將本實驗室所有的奴卡氏菌分離株進行分型所得知的結果，選擇10株脈衝型 ( pulsotype ) 及表現型各不同的菌株來進行魚隻抗奴卡氏菌分離株血清的製備。免疫過程分析第4、8、14週的血液及血清中其非特異性及特異性免疫試驗，第14週的NBT試驗上升的組別為93342、961113；第14週溶菌酶試驗上升的組別為93342、112及127三組。ELISA測定血清中抗體高低結果Ku040801及112兩組在第14週皆有上升。製備好的血清以西方墨漬法 ( western blot ) 來分析其血清中抗體對應的抗原，可發現抗體主要的辨識位置為29及72 kDa。最後自此10株分離株選擇961113分離株作為疫苗株進行疫苗製備。所製備的疫苗組別分別有961113超音波震碎不活化抗原、961113奈米不活化抗原、961113奈米不活化抗原混合FIA佐劑、961113奈米不活化抗原混合 ISA 763A佐劑、961113奈米不活化抗原混合 ISA 763A佐劑及熱休克蛋白，並以PBS免疫組作為對照組，觀察不同疫苗進行免疫後其特異性及非特異性免疫反應之比較，並且進行魚隻攻毒比較各組的保護效力。攻毒後第4週分析各組的相對保護力，分別為0、78%、22%、56%、33%，961113奈米不活化抗原免疫組別保護力與其他組別比較為最佳，其次為961113抗原混合ISA 763A佐劑。 關鍵字：奴卡氏菌、NBT、lysozyme、ELISA、西方墨漬法、奈米不活化抗原 【Abstract】 The phenotypic and genetic characterization of 92 isolated of fish pathogen Nocardia seriolae from sea water fish and fresh water fish, that were determined by pulse field gel electrophoresis (PFGE) and α-glucosidase activity by API ZYM kit. In PFGE analysis, digestions with restriction enzymes Xba I and Ase I produced 13 and 9 restriction pattern. In α-glucosidase activity, almost Taiwanese strains were positive, but 7 strains were no α-glucosidase activity. According to the result, 10 isolated strains were selected for anti-sera preparation and the formalin-killed bacteria were prepared. Largemouth bass were inoculated intraperitioneally with formalin-killed whole cell vaccine from 10 strains of N. seriolae and follow by secondary and third IP injection after primary immunization and secondary immunization. Increased nitro blue tetrazolium activity was observed in 2 strains at 14 weeks after third immunization. Increased lysozyme activity was observed in 3 strains at 14 weeks. The activity of specific immune response was monitored by an enzyme-linked immunosorbent assay and Western blot. The r