BSA-ISCOMs对欧洲鳗鲡免疫效应及抗细菌性病原的效果研究
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摘要
为证实免疫刺激复合物(ISCOMs)的非特异免疫增强作用,将牛血清白蛋白(BSA)制备成ISCOMs(BSA-ISCOMs).将欧洲鳗鲡(Anguilla anguilla)随机分为3组,分别用BSA-ISCOMs、BSA和磷酸盐缓冲液(PBS,对照组)腹腔注射免疫.二次免疫完成后第30d采血,测血清部分非特异性免疫指标,剩余鳗鲡分别用创伤弧菌(Vibrio vulnificus)和嗜水气单胞菌(Aeromona hydrophila)攻毒.结果显示,BSA-ISCOMs组的血清溶菌酶活力显著高于BSA组和对照组(p≤0.05);超氧化物歧化酶(SOD)和补体C3活力与对照组无显著差异(p>0.05).攻毒结果显示,BSA-ISCOMs组的存活率显著高于BSA组和对照组(p≤0.05),对创伤弧菌和嗜水气单胞菌攻击的相对免疫保护率分别为43.3%和51.7%.表明BSA-ISCOMs能有效增强欧洲鳗鲡对主要病原创伤弧菌和嗜水气单胞菌的抗病能力.
To test the effects of immune-stimulating complexes( BSA-ISCOMs),which was prepared by bovine serum albumin( BSA),on the non-specific immune responses of Anguillidae fish,European eels( Anguilla anguilla)were immunized intraperitoneally with BSA-ISCOMs or BSA,while the control group were injected with phosphate buffered solution( PBS). On the 30 th day after the second immunization,serum samples were collected for nonspecific immune parameters analysis,and the remaining eels were infected with Vibrio vulnificus and Aeromona hydrophila. The results showed that the eels immunized with BSA-ISCOMs presented significantly higher lysozyme activity than the eels injected with BSA or PBS( p≤0. 05). Superoxide dismutase( SOD) and complement component 3( C3) activities of the BSA-ISCOMs group were not significantly different from those of the BSA or PBS group( p > 0. 05). Survival rates of the BSA-ISCOMs group were significantly higher than those of BSA or PBS group( p≤0. 05),with a relative percent survival( RPS) of 43. 3% for V. vulnificus challenge and 51. 7% for A. hydrophila challenge. This study revealed the potential benefit of BSA-ISCOMs for the immune responses and disease resistance of European eel.
To test the effects of immune-stimulating complexes( BSA-ISCOMs),which was prepared by bovine serum albumin( BSA),on the non-specific immune responses of Anguillidae fish,European eels( Anguilla anguilla)were immunized intraperitoneally with BSA-ISCOMs or BSA,while the control group were injected with phosphate buffered solution( PBS). On the 30 th day after the second immunization,serum samples were collected for nonspecific immune parameters analysis,and the remaining eels were infected with Vibrio vulnificus and Aeromona hydrophila. The results showed that the eels immunized with BSA-ISCOMs presented significantly higher lysozyme activity than the eels injected with BSA or PBS( p≤0. 05). Superoxide dismutase( SOD) and complement component 3( C3) activities of the BSA-ISCOMs group were not significantly different from those of the BSA or PBS group( p > 0. 05). Survival rates of the BSA-ISCOMs group were significantly higher than those of BSA or PBS group( p≤0. 05),with a relative percent survival( RPS) of 43. 3% for V. vulnificus challenge and 51. 7% for A. hydrophila challenge. This study revealed the potential benefit of BSA-ISCOMs for the immune responses and disease resistance of European eel.
引文
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[2]Lamers C H,De H A,Van M U,et al.The reaction of the immune system of fish to vaccination:development of immunological memory in carp,Cyprinus carpio L.,following direct immersion in Aeromonas hydrophila bacterin[J].Journal of Fish Diseases,2006,8(3):253-262.
[3]Lan M Z,Peng X,Xiang M Y,et al.Construction and characterization of a live,attenuated esr B mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot,Scophthamus maximus(L.)[J].Fish&Shellfish Immunology,2007,23(3):521-530.
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[5]Morein B,Sundquist B,H9glund S,et al.Iscom,a novel structure for antigenic presentation of membrane proteins from enveloped viruses[J].Nature,1984,308(5 958):457-460.
[6]Morein B,Hu K F,Abusugra I.Current status and potential application of ISCOMs in veterinary medicine[J].Advanced Drug Delivery Reviews,2004,56(10):1 367-1 382.
[7]Morein B,L9vgren K,R9nnberg B,et al.Immunostimulating complexes[J].Clinical Immunotherapeutics,1995,3(6):461-475.
[8]Fossum C,Bergstrom M,Lovgren K,et al.Effect of iscoms and their adjuvant moiety(matrix)on the initial proliferation and IL-2 responses:comparison of spleen cells from mice inoculated with iscoms and/or matrix[J].Cellular Immunology,1990,129(2):414-425.
[9]Hsu S C,Schadeck E B,Delmas A,et al.Linkage of a fusion peptide to a CTL epitope from the nucleoprotein of measles virus enables incorporation into ISCOMs and induction of CTL responses following intranasal immunization[J].Vaccine,1996,14(12):1 159-1 166.
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[11]龚晖,林天龙,吴宗福,等.嗜水气单胞菌β-hem A-ISCOMs对鳗鲡的口服免疫效果[J].福建农业学报,2006,21(4):325-329.
[12]吴宗福,龚晖,陈红燕,等.嗜水气单胞菌重组β-hem A-ISCOMs对鳗鲡的浸泡免疫效果[J].水产学报,2007,31(3):374-378.
[13]龚晖,林天龙,张晓佩,等.含嗜水气单胞菌气溶素的免疫刺激复合物的制备及其对欧洲鳗的免疫效果[J].水产学报,2009,33(2):303-310.
[14]许斌福,林天龙,董传甫,等.鳗鲡创伤弧菌的分子鉴定[J].中国人兽共患病学报,2005,21(11):995-998.
[15]Morein B,Ekstr9m J,L9vgren K.Increased immunogeniccity of a non-amphipathic protein(BSA)after inclusion into ISCOMs[J].Journal of Immunological Methods,1990,128(2):177-181.
[16]Goldsmith C S,Miller S E.Modern uses of electron microscopy for detection of viruses[J].Clinical Microbiology Reviews,2009,22(4):552-563.
[17]Amend D F.Potency testing of fish vaccines[J].Developments in Biological Standardization,1981,49:447-454.
[18]L9vgren K,Morein B.The requirement of lipids for the formation of immunostimulating complexes(iscoms)[J].Biotechnology&Applied Biochemistry,1988,10(2):161-172.
[19]Kersten G F,Teerlink T,Derks H J,et al.Incorporation of the major outer membrane protein of Neisseria gonorrhoeae in saponin-lipid complexes(iscoms):chemical analysis,some structural features,and comparison of their immunogenicity with three other antigen delivery systems[J].Infection&Immunity,1988,56(2):432-438.
[20]Vinay T N,Park C S,Kim H Y,et al.Toxicity and dose determination of quillaja saponin,aluminum hydroxide and squalene in olive flounder(Paralichthys olivaceus)[J].Veterinary Immunology&Immunopathology,2014,158(1/2):73-85.
[21]Morein B.Iscoms[J].Veterinary Microbiology,1990,23(1/4):79-84.
[22]Mougin B,Bakouche O,Gerlier D.Humoral immune response elicited in rats by measles viral membrane antigens presented in liposomes and ISCOMs[J].Vaccine,1988,6(5):445-449.
[23]Sundquist B G,Czifra G,Stipkovits L.Protective immunity induced in chicken by a single immunization with Mycoplasma gallisepticum immunostimulating complexes(ISCOMS)[J].Vaccine,1996,14(9):892-897.
[24]Hgglund S,Hu K,Vargmar K,et al.Bovine respiratory syncytial virus ISCOMs-Immunity,protection and safety in young conventional calves[J].Vaccine,2011,29(47):8 719-8 730.
[25]Morein B,Hu K F,Abusugra I.Current status and potential application of ISCOMs in veterinary medicine[J].Advanced Drug Delivery Reviews,2004,56(10):1 367-1 382.
[26]汪开毓,陈霞,黄锦炉,等.无花果多糖对鲫鱼非特异性免疫功能的影响[J].水生生物学报,2011,35(4):630-637.
[27]Tang J,Cai J,Liu R,et al.Immunostimulatory effects of artificial feed supplemented with a Chinese herbal mixture on Oreochromis niloticus against Aeromonas hydrophila[J].Fish&Shellfish Immunology,2014,39(2):401-406.
[28]Zhu B,Liu G L,Gong Y X,et al.Single-walled carbon nanotubes as candidate recombinant subunit vaccine carrier for immunization of grass carp against grass carp reovirus[J].Fish&Shellfish Immunology,2014,41(2):279-293.
[29]Ellis A E.Immunity to bacteria in fish[J].Fish&Shellfish Immunology,1999,9(4):291-308.
[30]Ellis A E.Innate host defense mechanisms of fish against viruses and bacteria[J].Developmental&Comparative Immunology,2001,25(8/9):827-839.
[31]Grinde B,Lie,Poppe T,et al.Species and individual variation in lysozyme activity in fish of interest in aquaculture[J].Aquaculture,1988,68(4):299-304.
[32]Ackerman P A,Iwama G K,Thornton J C.Physiological and immunological effects of adjuvanted Aeromonas salmonicida vaccines on juvenile rainbow trout[J].Journal of Aquatic Animal Health,2000,12(2):157-164.
[33]Shailesh S,Sahoo P K.Lysozyme:an important defence molecule of fish innate immune system[J].Aquaculture Research,2008,39(3):223-239.
[34]张艳秋.油茶皂甙对罗非鱼生长、肉质和免疫功能影响及作用机理[D].杭州:浙江大学,2005.
[35]张声生,吴震宇,陈剑明,等.三七总皂苷改善高脂诱导脂肪肝大鼠模型氧化应激及胰岛素抵抗的研究[J].中国中西医结合杂志,2014,34(1):56-61.
[2]Lamers C H,De H A,Van M U,et al.The reaction of the immune system of fish to vaccination:development of immunological memory in carp,Cyprinus carpio L.,following direct immersion in Aeromonas hydrophila bacterin[J].Journal of Fish Diseases,2006,8(3):253-262.
[3]Lan M Z,Peng X,Xiang M Y,et al.Construction and characterization of a live,attenuated esr B mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot,Scophthamus maximus(L.)[J].Fish&Shellfish Immunology,2007,23(3):521-530.
[4]Guan R Z,Xiong J,Huang W S,et al.Enhancement of protective immunity in European eel(Anguilla anguilla)against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein[J].Acta Biochimica et Biophysica Sinica,2011,43(1):79-88.
[5]Morein B,Sundquist B,H9glund S,et al.Iscom,a novel structure for antigenic presentation of membrane proteins from enveloped viruses[J].Nature,1984,308(5 958):457-460.
[6]Morein B,Hu K F,Abusugra I.Current status and potential application of ISCOMs in veterinary medicine[J].Advanced Drug Delivery Reviews,2004,56(10):1 367-1 382.
[7]Morein B,L9vgren K,R9nnberg B,et al.Immunostimulating complexes[J].Clinical Immunotherapeutics,1995,3(6):461-475.
[8]Fossum C,Bergstrom M,Lovgren K,et al.Effect of iscoms and their adjuvant moiety(matrix)on the initial proliferation and IL-2 responses:comparison of spleen cells from mice inoculated with iscoms and/or matrix[J].Cellular Immunology,1990,129(2):414-425.
[9]Hsu S C,Schadeck E B,Delmas A,et al.Linkage of a fusion peptide to a CTL epitope from the nucleoprotein of measles virus enables incorporation into ISCOMs and induction of CTL responses following intranasal immunization[J].Vaccine,1996,14(12):1 159-1 166.
[10]Morein B,Villacrés-Eriksson M,Sj9lander A,et al.Novel adjuvants and vaccine delivery systems[J].Veterinary Immunology&Immunopathology,1996,54(1/4):373-384.
[11]龚晖,林天龙,吴宗福,等.嗜水气单胞菌β-hem A-ISCOMs对鳗鲡的口服免疫效果[J].福建农业学报,2006,21(4):325-329.
[12]吴宗福,龚晖,陈红燕,等.嗜水气单胞菌重组β-hem A-ISCOMs对鳗鲡的浸泡免疫效果[J].水产学报,2007,31(3):374-378.
[13]龚晖,林天龙,张晓佩,等.含嗜水气单胞菌气溶素的免疫刺激复合物的制备及其对欧洲鳗的免疫效果[J].水产学报,2009,33(2):303-310.
[14]许斌福,林天龙,董传甫,等.鳗鲡创伤弧菌的分子鉴定[J].中国人兽共患病学报,2005,21(11):995-998.
[15]Morein B,Ekstr9m J,L9vgren K.Increased immunogeniccity of a non-amphipathic protein(BSA)after inclusion into ISCOMs[J].Journal of Immunological Methods,1990,128(2):177-181.
[16]Goldsmith C S,Miller S E.Modern uses of electron microscopy for detection of viruses[J].Clinical Microbiology Reviews,2009,22(4):552-563.
[17]Amend D F.Potency testing of fish vaccines[J].Developments in Biological Standardization,1981,49:447-454.
[18]L9vgren K,Morein B.The requirement of lipids for the formation of immunostimulating complexes(iscoms)[J].Biotechnology&Applied Biochemistry,1988,10(2):161-172.
[19]Kersten G F,Teerlink T,Derks H J,et al.Incorporation of the major outer membrane protein of Neisseria gonorrhoeae in saponin-lipid complexes(iscoms):chemical analysis,some structural features,and comparison of their immunogenicity with three other antigen delivery systems[J].Infection&Immunity,1988,56(2):432-438.
[20]Vinay T N,Park C S,Kim H Y,et al.Toxicity and dose determination of quillaja saponin,aluminum hydroxide and squalene in olive flounder(Paralichthys olivaceus)[J].Veterinary Immunology&Immunopathology,2014,158(1/2):73-85.
[21]Morein B.Iscoms[J].Veterinary Microbiology,1990,23(1/4):79-84.
[22]Mougin B,Bakouche O,Gerlier D.Humoral immune response elicited in rats by measles viral membrane antigens presented in liposomes and ISCOMs[J].Vaccine,1988,6(5):445-449.
[23]Sundquist B G,Czifra G,Stipkovits L.Protective immunity induced in chicken by a single immunization with Mycoplasma gallisepticum immunostimulating complexes(ISCOMS)[J].Vaccine,1996,14(9):892-897.
[24]Hgglund S,Hu K,Vargmar K,et al.Bovine respiratory syncytial virus ISCOMs-Immunity,protection and safety in young conventional calves[J].Vaccine,2011,29(47):8 719-8 730.
[25]Morein B,Hu K F,Abusugra I.Current status and potential application of ISCOMs in veterinary medicine[J].Advanced Drug Delivery Reviews,2004,56(10):1 367-1 382.
[26]汪开毓,陈霞,黄锦炉,等.无花果多糖对鲫鱼非特异性免疫功能的影响[J].水生生物学报,2011,35(4):630-637.
[27]Tang J,Cai J,Liu R,et al.Immunostimulatory effects of artificial feed supplemented with a Chinese herbal mixture on Oreochromis niloticus against Aeromonas hydrophila[J].Fish&Shellfish Immunology,2014,39(2):401-406.
[28]Zhu B,Liu G L,Gong Y X,et al.Single-walled carbon nanotubes as candidate recombinant subunit vaccine carrier for immunization of grass carp against grass carp reovirus[J].Fish&Shellfish Immunology,2014,41(2):279-293.
[29]Ellis A E.Immunity to bacteria in fish[J].Fish&Shellfish Immunology,1999,9(4):291-308.
[30]Ellis A E.Innate host defense mechanisms of fish against viruses and bacteria[J].Developmental&Comparative Immunology,2001,25(8/9):827-839.
[31]Grinde B,Lie,Poppe T,et al.Species and individual variation in lysozyme activity in fish of interest in aquaculture[J].Aquaculture,1988,68(4):299-304.
[32]Ackerman P A,Iwama G K,Thornton J C.Physiological and immunological effects of adjuvanted Aeromonas salmonicida vaccines on juvenile rainbow trout[J].Journal of Aquatic Animal Health,2000,12(2):157-164.
[33]Shailesh S,Sahoo P K.Lysozyme:an important defence molecule of fish innate immune system[J].Aquaculture Research,2008,39(3):223-239.
[34]张艳秋.油茶皂甙对罗非鱼生长、肉质和免疫功能影响及作用机理[D].杭州:浙江大学,2005.
[35]张声生,吴震宇,陈剑明,等.三七总皂苷改善高脂诱导脂肪肝大鼠模型氧化应激及胰岛素抵抗的研究[J].中国中西医结合杂志,2014,34(1):56-61.