2017, 41(4): 622-627. doi: 10.11964/jfc.20160310301
关键词： 虹鳟 , 植物乳杆菌 , IPNV VP2-VP3 , 口服疫苗 , 免疫程序
Keywords： Oncorhynchus mykiss , Lactobacillus plantarum , IPNV VP2-VP3 , oral vaccine , immunization program
为比较鱼源乳酸菌表达系统口服疫苗在不同免疫程序下诱导鱼免疫应答水平的差异，确定鱼源乳酸菌表达系统口服免疫虹鳟幼鱼的免疫程序。本研究构建了重组表达IPNV VP2-VP3蛋白的鱼源植物乳杆菌L1212，将重组菌pPG612-VP2-VP3/L1212包裹颗粒饲料，口服免疫虹鳟幼鱼。免疫程序分为连续免疫组、免疫1 d后间隔1 d再免疫1 d组、连续免疫4 d后32 d加强免疫1次组和间隔免疫后32 d加强免疫1次组。免疫后进行尾动脉采血，间接ELISA方法检测各组血清抗体效价，免疫接种66 d后，腹腔注射IPNV，计算各组相对免疫保护率。确定颗粒饲料按照1 mL/g比例与108 CFU/mL重组菌pPG612-VP2-VP3/L1212和2.5%海藻酸钠混合，于20℃烘干制备口服疫苗。间隔免疫组血清抗体效价和攻毒保护率均显著高于其他组，并且免疫2次的间隔免疫组的血清抗体效价和攻毒保护率均显著高于免疫1次间隔免疫组。采用重组菌包被颗粒饲料饲喂虹鳟幼鱼，间隔免疫的免疫程序和免疫保护率优于连续免疫的免疫程序，对IPNV的入侵起到保护作用。
Comparison of the expression of fish-derived lactic acid bacteria expression system in oral vaccines was made under different immune process, to determine fish source Lactobacillus expression system immune program for immunization of juvenile rainbow trout. In this study, we constructed L. plantarum L1212 in recombinant protein expression IPNV VP2-VP3, then the recombinant strain pPG612-VP2-VP3/ L1212 was coated with pellet feed and immune Oncorhynchus mykiss juvenile fish. Immunization program was divided into continuous immunization group: the group that is immune for a single day after immunization for 1 day; the group that strengthens the immune 1 times after 4 days of continuous immunization 32 d and the group that strengthens the immune 1 times after interval immunization 32 d. After immunization, tail artery blood collection is carried out, and the serum antibody titers are measured by indirect ELISA. After immunization for 66 days, IPNV is injected abdominal cavity and the relative immune protection rates were calculated for each group. To determine the pellet feed in accordance with 1 mL/g ratio and 108 CFU/mL recombinant bacteria pPG612-VP2-VP3/ L1212 and 2.5% sodium alginate mixed 20℃ drying preparation of oral vaccine; the titer and challenge protection rate of serum antibody in interval immunization group were significantly higher than those of other groups, and the serum antibody titer and challenge protection rate in the twice-immunization group were significantly higher than those of the group immunized once. The immunization program and the immunoprotection rate of the juvenile O. mykiss were collected by the recombinant bacteria. The immunization program was superior to the immunization program for continuous immunization and protected the invasion of IPNV.
| [1] | 夏春. 水生动物疾病学[M].北京:中国农业大学出版社, 2005. Xia C. Aquatic Animal Diseases[M]. Beijing:China Agricultural University Press, 2005(inChinese). |
| [2] | 刘兴发.鱼传染性胰腺坏死[J]. 中国兽医杂志, 1998, 24(1):51-53. Liu X F. Infectious pancreatic necrosis of fish[J]. Chinese Journal of Veterinary Medicine, 1998, 24(1):51-53(in Chinese). |
| [3] | Romalde J L, Luzardo-Alvárez A, Ravelo C, et al. Oral immunization using alginate microparticles as a useful strategy for booster vaccination against fish lactoc-cocosis[J]. Aquaculture, 2004, 236(1-4):119-129. |
| [4] | Shin S P, Gomez D K, Kim J H, et al. Detection and genetic analysis of aquabirnaviruses in subclinically infected aquarium fish[J]. Journal of Veterinary Di-agnostic Investigation, 2011, 23(2):325-329. |
| [5] | Munang'andu H M, Sandtrø A, Mutoloki S, et al. Immunogenicity and cross protective ability of the central VP2 amino acids of infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar L.)[J]. PLoS One, 2013, 8(1):e54263. |
| [6] | Park J W, Jeong G. Identification of VP3 as an important neutralising epitope from DRT strain, a Korean isolate of infectious pancreatic necrosis virus (IPNV)[J]. Fish & Shellfish Immunology, 1996, 6(3):207-219. |
| [7] | Moon C H, Do J W, Cha S J, et al. Comparison of the immunogenicity of recombinant VP2 and VP3 of in-fectious pancreatic necrosis virus and marine birnavirus[J]. Archives of Virology, 2004, 149(10):2059-2068. |
| [8] | Ballesteros N A, Saint-Jean S R, Perez-Prieto S I. Immune responses to oral pcDNA-VP2 vaccine in relation to infectious pancreatic necrosis virus carrier state in rainbow trout Oncorhynchus mykiss[J]. Veterinary Immunology and Immunopathology, 2015, 165(3-4):127-137. |
| [9] | Zhao L L, Liu M, Ge J W, et al. Expression of infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts[J]. Vaccine, 2012, 30(10):1823-1829. |
| [10] | Liu M, Zhao L L, Ge J W, et al. Immunogenicity of Lactobacillus-expressing VP2 and VP3 of the infectious pancreatic necrosis virus (IPNV) in rainbow trout[J]. Fish & Shellfish Immunology, 2012, 32(1):196-203. |
| [11] | 刘敏, 张英, 张琳琳, 等.虹鳟肠道植物乳杆菌的分离及特性研究[J]. 东北农业大学学报, 2014, 45(12):24-30. Liu M, Zhang Y, Zhang L L, et al. Isolation and characterization of indigenous Lactobacillus plantarum from rainbow trout intestine[J]. Journal of Northeast Agricultural University, 2014, 45(12):24-30(in Chinese). |
| [12] | Dadar M, Memari H R, Vakharia V N, et al.Protective and immunogenic effects of E.coli-expressed infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in rainbow trout[J]. Fish & Shellfish Immunology, 2015, 47(1):390-396. |
| [13] | Kai Y H, Chi S C. Efficacies of inactivated vaccines against betanodavirus in grouper larvae (Epinepheluscoioides) by bath immunization[J]. Vaccine, 2008, 26(11):1450-1457. |
| [14] | Sinyakov M S, Dror M, Lublin-Tennenbaum T, et al.Nano-and microparticles as adjuvants in vaccine design:success and failure is related to host natural antibodies[J]. Vaccine, 2006, 24(42-43):6534-6541. |
| [15] | Altun S, Kubilay A, Ekici S, et al.Oral vaccination against lactococcosis in rainbow trout (Oncorhynchus mykiss) using sodium alginate and poly (lactide-co-glycolide) carrier[J]. Kafkas Üniversitesi Veteriner Fakultesi Dergisi, 2010, 16(2):211-217. |
| [1] | 陈晶, 聂青, 刘妍. 《WHO基本药物示范目录》与我国《国家基本药物目录》动态调整程序比较与借鉴.水产学报,2015(3): 289-293.doi:10.3866/PKU.WHXB201503022 |
