斑点叉尾鮰血清免疫球蛋白的提取纯化及部分特性分析
|
摘要
本文通过饱和硫酸铵分步盐析结合Sepharose-4B凝胶柱以及DEAE—52阴离子交换柱层析分别提取纯化非免疫和温和气单胞菌免疫的斑点叉尾鮰血清免疫球蛋白(Ig)，采用单克隆抗体(MAF-13株)结合酶联免疫吸附(ELISA)检测提取物中血清免疫球蛋白的活性。结果表明非免疫斑点叉尾鮰的血消Ig主要存在于35％～45％的硫酸铵沉淀区间，而免疫斑点叉尾鮰的血清Ig则分布在35％～55％的硫酸铵沉淀区间内。在Sepharose-4B凝胶柱和DEAE—52阴离子交换柱层析纯化中Ig均出现在第一个蛋白峰。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行纯度鉴定，结果发现经过系列提纯的Ig样品杂蛋白条带逐渐减少，DEAE—52纯化后的Ig仅在重、轻链位置出现明显条带，表明纯化后的血清免疫球蛋白纯度较高。纯化的血清Ig用以制备兔抗斑点叉尾鮰血清Ig的多克隆抗体．采用ELISA方法分别测定单克隆抗体和多克隆抗体的特异性，结果表明抗斑点叉尾鮰血清Ig的多克隆抗体和单克隆抗体均与欧洲鳗鲡和日本鳗鲡有免疫交叉反应。应用变性还原(denatured and reduced)、变性非还原(denatured but non-reduced)和非变性非还原条件下(non-denatured and non-reduced)的免疫印迹(western blot)试验对纯化的Ig进行结构分析，以抗斑点叉尾鮰血清Ig的单克隆抗体和多克隆抗体碱性磷酸酶标记物为探针进行检测。结果表明免疫斑点叉尾鮰血清Ig的重链分子量为72KD，轻链有三条，分子量分别为21KD、23.5KD利26KD；而非免疫斑点叉尾鮰血清Ig中发现有2条重链，分子量分别为72KD和55KD。梯度PAGE和Western blot测得非还原非变性条件下免疫球蛋白的分子量约为900KD。在SDS变性非还原梯度PAGE和Western blot中发现血清Ig出现多种结构形式，分子量分别为870KD、760KD、525KD、330KD利230KD。
Serum immunoglobulin (Ig) from normal and Aeromonas sobria (A.s) immunized channel catfish (Ictalurus punctatus) were each purified by using ammonium sulfate precipitation followed by sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. The purified Ig fractions were defined by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (MAF-13) before being pooled and accessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that normal channel catfish serum Ig were mainly present in the range of 35% - 45% ammonium sulfate precipitation, however, the serum Ig of immunized channel catfish were present in the range of 35%-55% ammonium sulfale precipitation. The immunoglobulin appeared in the first protein peak both in the Sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. SDS-PAGE results showed that serum Ig had been further purified by each step of purification. The purified Ig were used as antigen to produce rabbit antiserum. Polyclonal and monoclonal antibodies both reacted with serum of European eel (Anguilla anguilla) and Japanese eel (Anguilla Japonica) in ELISA. Structure analysis of serum Ig were performed by Western blot under denatured and reduced, denatured but non-reduced, non-denatured and non-reduced conditions using monoclonal and polyclonal antibodies as probes. The results showed that the denatured and reduced serum Ig of normal channel catfish had two different heavy (H) chains with molecular weight about 72 0 00 and 55 000 respectively. For the immunized channel catfish, however, the molecular weight of heavy chain was predominantly determined to be 72 000, and three distinct light (L) chains were found with molecular weight of 21 000, 23 500, 26 000 respectively. The naive serum Ig was estimated to have a molecular weight about 900KD using 3%~12% gradient PAGE and western blot under non-reduced and non-denatured conditions. This polymeric Ig could dissociated into several subpopulations with molecular weight about 870 000,760 000,525 000,330 000 and 230 000 when analyzed in the present of denaturing solvent (SDS).
Serum immunoglobulin (Ig) from normal and Aeromonas sobria (A.s) immunized channel catfish (Ictalurus punctatus) were each purified by using ammonium sulfate precipitation followed by sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. The purified Ig fractions were defined by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (MAF-13) before being pooled and accessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that normal channel catfish serum Ig were mainly present in the range of 35% - 45% ammonium sulfate precipitation, however, the serum Ig of immunized channel catfish were present in the range of 35%-55% ammonium sulfale precipitation. The immunoglobulin appeared in the first protein peak both in the Sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. SDS-PAGE results showed that serum Ig had been further purified by each step of purification. The purified Ig were used as antigen to produce rabbit antiserum. Polyclonal and monoclonal antibodies both reacted with serum of European eel (Anguilla anguilla) and Japanese eel (Anguilla Japonica) in ELISA. Structure analysis of serum Ig were performed by Western blot under denatured and reduced, denatured but non-reduced, non-denatured and non-reduced conditions using monoclonal and polyclonal antibodies as probes. The results showed that the denatured and reduced serum Ig of normal channel catfish had two different heavy (H) chains with molecular weight about 72 0 00 and 55 000 respectively. For the immunized channel catfish, however, the molecular weight of heavy chain was predominantly determined to be 72 000, and three distinct light (L) chains were found with molecular weight of 21 000, 23 500, 26 000 respectively. The naive serum Ig was estimated to have a molecular weight about 900KD using 3%~12% gradient PAGE and western blot under non-reduced and non-denatured conditions. This polymeric Ig could dissociated into several subpopulations with molecular weight about 870 000,760 000,525 000,330 000 and 230 000 when analyzed in the present of denaturing solvent (SDS).
引文
1．王长法，安利国，杨桂文等．鱼类免疫球蛋白研究进展．中国水产科学，1999，6(2)：105～107
2．刘秀梵主编．单克隆抗体在农业上的应用．安徽科学技术出版社，1994
3．曲凌云，孙修勤，战文斌等．牙鲆抗淋巴囊肿病毒免疫球蛋白的纯化．高技术通讯，2000，9：14～18
4．江育林，李燕，于平．草鱼免疫应答的初步研究．水生生物学报，1991，15(4)：321～326
5．邬国民．斑点叉尾鮰的养殖现状和前景展望．珠江水产，1998，23：16～20
6．吴婌勤．一种新的斑点叉尾鮰出血病．鱼类病害研究，1992，1
7．张永安，聂品．鳜血清免疫球蛋白的分离纯化及其亚单位分子量的测定．水生生物学报，1998，22(2)：192～194
8．李亚南，陈全震，邵健忠等．动物学研究，1995，16(1)：83～93
9．杨先乐．鱼类免疫学研究的进展．水产学报，1989，13(3)：271～284
10．杨桂文，安利国，王长法．鲤肠粘液与血清中免疫球蛋白的比较研究．中国水产科学，6(4)：109～110
11．杨桂文，安利国，王长法．鲤鱼皮肤粘液与血清中免疫球蛋白的比较研究．动物学研究，1998，19(6)：489～492
12．杨桂文，安利国，温武军等．鲤胆汁与血清中免疫球蛋白的比较研究．水产学报，1998，22(3)：199～203
13．陈昌福，纪国良，罗宇良等．草鱼体表和肠粘液中免疫球蛋白的初步分析．鱼病学研究论文集，1999，2：21～25
14．陈昌福．用直接荧光抗体法对鲤血液中淋巴细胞类型的初步鉴定．华中农业大学学报，1998，17(2)，154～158
15．周丽，战文斌，宋微波等．指状拟舟虫诱导牙鲆抗血清免疫球蛋白分析．中国水产科学，2000，7：28～31
16．孟凡国，李亚南．鱼类体液免疫研究进展．国外兽医学—畜禽疾病，1996，17(4)：16～18
17．林天龙，陈强，俞伏松等．欧洲鳗血清免疫球蛋白纯化及部分特性分析．水产学报，2000，25(1)：52～57
18．赵振山．沟鲶养殖．科学出版社，1994
19．钟明超，林浩然．硬骨鱼类胚胎和胚后发育阶段的免疫机制．中国水产科学，1996．3(2)：97～102
20．夏春，川合研儿，楠田理一．鳗鲡淋巴细胞表面存在不同表型的免疫球蛋白．水产学报，1996，20(4)：361～364
21．聂品．鱼类非特异性免疫研究的新进展．水产学报，1997，21(1)：69～73
22．郭尧君．蛋白质电泳实验技术．北京：科学出版社，1998
23．葛雷，常洪．斑点叉尾鮰小瓜虫病的防治．鱼类病害研究，1996，4：24
24．葛雷，常洪．斑点叉尾鮰 烂尾病的初步研究．鱼类病害研究，1996，4：23
25. Amemjya C T, Litmen G W. Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunogobulin gene organization in a primative teleost species. Proc Natl Acad Sci USA, 1990, 87: 811～815
26. Bang J D, Kim J W, Lee S D, et al. Humoral immune response of flounder to Edwardsiella tarda: the presence of various sizes of immunoglobulins in flounder. Dis Aqu Org, 1996, 26: 197～203
27. Bly J E, Grimm A S, Morris I G, et al. Transfer of passive immunity from mother to young in a teleost fish: Hemagglutinating activity in the serum and eggs of plaice, Pleuronectes platessa. L. Comp Biochem Pnysiol. 1986, 84A: 309～313
28. Bourmand C A F, Romestand B, Bouix G. Isolation and partial characterization of IgM-like seabass (Dicentrarchus labrax L.1758) immunoglobulins. Aquaculture, 1995, 132: 53～58
29. Bradshaw C M, Clem L M, Sigel M M. IgM antibodies in fish mucus. Proc Soc Exp Biol Med. 1971, 136: 1122
30. Chen changfu, Li Jing, Riichi Kusuda. Preliminary study on maternal immunity of grass carp (Ctenopharyngodon idellus). Journal of Huazhong Agricultural University. 1996,15(1),73～77
31. Clem L W et al. Evolution of lymphocyte subpopulations their interactions and temperature sensitivities. In: Warr G M, Cohen N. Ed. Phylogenesis of Immune Function. Boca Raton, Florida: CRC Press. 1991, 191～214
32. Clem L W. Phylogeny of immunoglobulin structure and function. Ⅳ. Immunoglobulins of the giant grouper, Epinephelus itaira, d Biol Chem Biological, 1971, 246: 9～15
33. Davidson G A et al. Route of immunization influences the generation of antibody secreting cells in the gut of rainbow trout (Oncorhynchus mykiss). Dev Comp Immunol, 1993, 17: 373～376
34. Davidson G A. Aspects of mucosal immunity in rainbow trout, Oncorhynchus mykiss(Walbaum 1792). PhD thesis, Aberdeen University. 1991.
35. Deluca D, Willson M, Warr G W et al. Lymphocyte heterogeneity in the trout, Salmo gairdneri, defined with monoclonal antibodies to IgM. Eur J Immunol, 1983, 13: 546～551
36. Dorson M. Applied immunology of fish. In: Symposium on fish vaccination (Ed:by De Kinkilin, P). 1984, 39～74.
37. Elcombe B M, Chang R J, Taves C J, et al. Evolution of antibody structure and effector functions: comparative hemolytic activities of monomeric and tetrameric IgM from rainbow trout, Salmo gairdneri. Comp Biochem Physiol, 80B: 697～706
38. Ellis E et al. Surface immunoglobulins on the lymphocytes of the skate Reja naevua. Eur J Immunol, 1975, 5: 726～728
39. Estevez J et al. Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays. Vet Immunol Immunopathol, 1994, 41: 353～366
40. Estevez J, Leiro J, Sanmartin M L, et al. Isolation and partial characterization of turbot (Scophthalmus maximus) immunoglobulins. Comp Biochem Physiol, 1993,1 05A: 275～281
41. Fletcher T C, Grant P T. Immunoglobulin in the serum and mucus of the plaice(Pleuronectes platessa),J Biochem, 1969, 115: 65
42. Fuda H, Hara Aj, Yamazaki F, et al. A speculiar immunoglobulin M (IgM) identified in eggs of chum salmo (Oncorhynchus keta). Dev Comp Immunol, 1992, 16: 415～423
43. Fuller L, Murray J, Jensen J A. Isolation from nurse shark serum or immune 7S antibodies with two different molecular weight H-chains. Immunochemistry, 1978, 15: 251～259
44. Ghaffari S H, Lobb C J. Cloning and sequence analysis of channel catfish heavy chain cDNA indicate phylogenetic diversity within the IgM immunoglobulin family. J Immunol, 1989, 142: 1356～1365
45. Ghaffari S H, Lobb C J. Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogenety of Ig heavy chains, J Immunology, 1989, 143: 2730～2739
46. Graham S, Secombes C J. Cellular requirements for lymphocykine secretion by rainbow trout Salmo gairdneri leucocytes. Dev Comp Immunol, 1990, 14: 59～68
47. Hagiwara k, Kobayashi K, Kajii T, et al. J-chain-like component in 18-S immunoglobulin of the skate Raja Kenojei. a cartilaginous fish. Mol Immunol, 1985, 22(7): 775～778
48. Harding F A, Cohen N, Litman G W. Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea. Nucleic Acids Res, 1990, 18(4): 1015～1020
49. Harding F A,Amemiya C T. Two distinct immunolobulin heavy chain isotypes in a primative, cartilaginous fish, Raja erinacea. Nucleic Acids Res, 1990, 18(21): 6369～6376.
50. Harlow, David Lane. Antibodies-A laboratory manual. Cold Spring Harbor Laboratory Press, NY, 1988
51. Harrell L W, Etlinger L M, Hodgins H O. Humoral factors important in resistance of salmonid fish to bacterial disease. Ⅱ. Anti-Vibro anguillarum activity in mucus and observations on complement. Aquaculture, 1976, 7: 363～370
52. Hart S, Warthmell A B, Harris J E, et al. Gut iummunology in fish: a review. Dev Comp Immunol, 1988, 12: 453～480
53. Havarstein L S, Assjord P M, Ness S, et al. Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar). Dev Comp Immunol, 1988, 12: 773～785
54. Hayman J R, Lobb C J. Immunoglobulin in the eggs of the channel catfish (Ictalurus punctatus). Dev Comp Immunol, 1993, 17: 241～248
55. Hung H W, Lo C T, Tseng C C, et al. Humoral immune response of Japanese eel, Anguilla japonica, to Pleistophora anguillarum Hoshina 1951(Microspora). J Fish Dis, 1996, 19: 243～250
56. Itami T et al. Purification and characterization of immunoglobulin in skin mucus and serum of ayu. Nippon Suisan Goakkaish, 1988, 54: 1611～1617
57. Jurd R D. Specialization in the teleost and anuran immune response. In: M J Manning, M F Tatner. A comparative critique in fish immunology. London: Academic Press. Inc, 1985, 9～28
58. Kobayashi K, Tomonaga S, Kajii T. A second class of immunoglobulin other than IgM present in the serum of a cartilaginous fish, the skate, Raja Kenjei: isolation and characterization. Mol Immunol, 1984, 21: 397～404.
59. Kobuku G, Litman R, Shambiott M J, et al. Diverse organization of immunoglobulin VH gene local in a Primitive vertebrate. EMBO, 1988, 7: 3413～3422
60. Laemmli U K. Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature(London), 1970, 227: 680～685
61. Lobb C J, Clem L W. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev Comp Immunol, 1982, 6: 473～479
62. Lobb C J, Clem L W. Immunoglobulin light chain classes in a teleost fish. J Immunol, 1984, 132: 1917～1923
63. Lobb C J, Clem L W. Phylogeny of immunoglobulin structure and function.X.Humoral immunoglobulins of the sheepshead, Archosargus probatocephalus. Dev Comp Immunol, 1981c, 5: 271～282
64. Lobb C J, Clem L W. The metabolic relationships of the immunoglobulins in fish serum, cutaneous mucus, and bile. J Immunol. 1981,127:1525
65. Lobb C J, Olson M O J. Immunoglobulin heavy H chain isotypes in a teleost fish. J Immunol, 1988, 141: 1236～1245
66. Lobb C J. Secretary immunity induced in catfish, Ictalurus punctatus, following bath immunization. Dev Comp Immunol, 1987, 11: 727～738
67. Lobb C J. Structural diversity of channel catfish immunoglobulins. Veterinary Immunology and Immunopathology, 1986, 12: 7～12
68. Loghothetic P N, Austin B, Immune response of rainbow trout (Oncorhynchus mykiss, Walbaum) to Aeromonas hydrophila. Fish Shellfish Immunol. 1994, 4: 239～254
69. Lumsden J S et al. Detection of a distinct gill-surface antibody response following horizontal infection and bath challenge of brook trout Salvelinus fontinalis with Flavobacterium branchiophilum, the causative agent of bacterial gill disease. Dis Aqua Org, 1993, 16: 21～27
70. Manning M J. Fishes. In: Tumer R J ed. Immunology comparative approach. Britain: John Wiley & Sons Ltd. 1994, 69～100
71. Mccumber L J, Clem L W. A comparative study of J chain: structure and stoichicometry in human and nurse shark IgM. Immunochemistry, 1976, 13, 479～484
72. McGhee J R, Mestecky J, Derzbaugh M J, et al. The mucosal immune system from fundamental concepts to vaccine development. Vaccine, 1992 10: 75～88
73. Mestecky J, Kulhavy R, Schrohenloher R E, et al. Identification and properties of J chain isolated from catfish macroglobulin. J Immunol, 1975, 115: 993～997
74. Mor A, Aviation R R. Evidence of transfer of immunity from mother to eggs in tilapias. Israeli J. Aquacult(Bamidgeh), 1988, 40: 22～28
75. Mor A, Avtalion R R. Transfer of antibody activity from immunized mother to embryo in tilapias, J Fish Biol. 1990, 37: 249～255
76. Nakao M, Moritomo T, Tomana M, et al. Isolation of cDNA encoding the constant region of the immunoglobulin heavy-chain from common carp. (Cyprinus carpio L.). Fish Shellfish Immunol, 1998, 8(6):425～434
77. Navarro V et al. Immuno(cyto)chemical characterization of monoclonal antibodies to githead seabream (Sparus aurata) immunoglobulin. Fish Shellfish Immunol, 1993, 3: 167～177
78. Nossal G J V, Sberg A A, GL,Austin C M. Single cell studies on 19s antibody production. J. Exp Med, 1964, 119: 485～502
79. Ourth D D. Purification and quantification of channel catfish (Ictalurus punctatus) immunoglobulin M. douranl of Applied Ichthyology, 1986, 3: 140～143
80. Phillips J O, Ourth D D. Isolation and molecular weight determination of two immunoglobulin heavy chains in the channel catfish, Ictalurus punctatus. Comparative Biochemistry and Physiology, 1986, 85B: 49～54
81. Pilstrom L, Peterson A. Isolation and partial characterization of immunoglobulin from aye (Gadus morhua L.). Dev Comp Immunol, 1991, 15: 143～152
82. Rabbits T H, Forster A, Dunmik W, Bentley D L. The role of gene deletion in the immunglobulin heavy chain switch. Nature, 1980, 283: 351～356.
83. Rombout J H W M, et al. Differences in mucus and serum immunoglobulin of carp (Cyprinius carpio L). Dev Comp Immunol, 1993, 17: 309～317
84. Rombout J H W M, Bot H E, Lamers C H J, et al. Immunological importance of the second gut segment of carp. Ⅱ. Characterization of mucosal leucocytes. J Fish Biol, 1989a, 35: 167～178
85. Rombout J H W M, Van den Berg A A, Van den Berg C T GS,et al. Immunological importance of the second gut segment of carp. Ⅱ. System and/or mucosal immune responses after immunization with soluble or particulate antigens. J Fish Biol, 1989b, 35: 179～186
86. Rombout J W H M, Blok L J, Lamers C H J, et al. Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system. Dev Comp Immunol, 1986, 10: 341～351
87. Sanchez C, Domiguez J, Coll J. Immunoglobulin heterogeneity in the rainbow trout, Salmo gairdneri Richardson. J Fish Dis, 1989, 12: 459～465
88. Sanchez C, Dominguez J. Trout immunoglobulin populations differing in light chains revealed by monoclonal antibodies. Mol Immunol, 1991, 28: 1271～1277
89. Sanchez C, Lopez-Fierro P, et al. Characterisation of monoclonal antibodies against heavy and light chains of trout immunoglobulin. Fish Shellfish Immunology, 1993, 3(4): 237～251
90. Scapigliati G, Romano N, et al. Monoclonal antibodies against sea bass Dicentrarchus labrax L. Immunoglobulins: immunolocalisation of immunoglobulin-bearing cells and applicability in immunoassays. Fish Shellfish Immunology, 1996,6(5):383～401
91. Secombes C J et al. The development of an ELISPOT assay to quantify total and specific antibody-secreting cells in dab Limanda limanda L. Fish Shellfish Immunol, 1991, 1: 87～97
92. Secompes C J et al. Separation of lymphocyte subpopulation in carp (Cyprinus carpio L) by monoclonal antibodies immunohistochemical studies. Immunol,1983, 48: 165～175
93. Service R F. Triggering the first line of defense. Science, 265: 1522～1524
94. Shelton E, Smith M. The ultrastucture of carp (Cyprinus carpio) immunoglobulin: a tetrameric Immunogllbulin. J. Molec. Biol. 1970, 54, 615～617
95. Smith S A, Gebhard D H, Housman J M et al. Isolation, purification and molecular-weight determination of serum immunoglobulin from Oreochromis aureus. Journal of Aquatic Animal Health, 1993, 5: 23～35
96. St Louis-Cormier E A, Osterland C K, Aderson P D et al. Evidence for a cutaneous secretory immune system in the rainbow trout (Salmo gairdneri). Dev Comp Immunol, 1984, 8:71
97. Takahashi Y, Kawahara E. Maternal immunity in newborn fry of the ovoviparous guppy. Nippon Suisan Gakkaishi, 1987,53:721～735
98. Takemura A. Changes in an immunoglobulin M(IgM)-like protein during larval stages in tilapia, Oreochromis mossambicus. Aquaculture, 1993,115:233～241
99. Thuvander A,Fossum C, Lorenzen N. Monocolnal antibodies to salmonid immunoglobulin: characterization and applicability to immunoassays. Dev Comp Immunol. 1990,14:415～423
100. Tomanaga S et al. Characterization of serum immunoglobulins in a chendrostean fish. Dev Comp Immunol.1993, 17: 515～524
101. Trump G N et al. Antibody response of goldfish to bovine serum albumin: Primary and secondary responses. Immunology, 1970,19:627
102. Tucker C S, Robinson E H. Channel catfish farming handbook. Elsevier Science Publishers. 1991.
103. Warr G W et al. Phylogenetic origins of immune recognition: lymphocyte surface immunoglobulin of the goldfish, carassius auratus. Proc Nat Acad Sci. 1976, 73: 2476～2480
104. Whittington K J. Purification and partial characterization of serum immunoglobulin of the European perch (Perca fluviatilis L.). Fish Shellfish Immunol, 1993, 3: 331-343
105. Wilson M R, Warr G W. Fish immunoglobulins and the genes that encode them. Annual Review of Fish Diseses. 1992, 2: 201～221
106. Wolf K, Quimby M C, Bradford A D, et al. Egg-associated transmission of IPN virus of trout. Virology, 1963, 21: 317～321
107. Xia C et al. Presence of mitogen receptors on the lymphocyte of eel, Anguilla japonica. Suisanzoshoku, 1994b, 42: 53～56
108. Xia C et al. Receptors for sheep red blood cells, imunoglobulin M and complement detected on the lymphocyte of eel, Anguilla japonica. Suisanzoshoku, 1994a, 42: 47～51
109. Xia C et al. Studies on the heterogeneity of lymphocyte population in eel, Anguilla japonica. Suisanzoshoku, 1993, 41: 119～123
2．刘秀梵主编．单克隆抗体在农业上的应用．安徽科学技术出版社，1994
3．曲凌云，孙修勤，战文斌等．牙鲆抗淋巴囊肿病毒免疫球蛋白的纯化．高技术通讯，2000，9：14～18
4．江育林，李燕，于平．草鱼免疫应答的初步研究．水生生物学报，1991，15(4)：321～326
5．邬国民．斑点叉尾鮰的养殖现状和前景展望．珠江水产，1998，23：16～20
6．吴婌勤．一种新的斑点叉尾鮰出血病．鱼类病害研究，1992，1
7．张永安，聂品．鳜血清免疫球蛋白的分离纯化及其亚单位分子量的测定．水生生物学报，1998，22(2)：192～194
8．李亚南，陈全震，邵健忠等．动物学研究，1995，16(1)：83～93
9．杨先乐．鱼类免疫学研究的进展．水产学报，1989，13(3)：271～284
10．杨桂文，安利国，王长法．鲤肠粘液与血清中免疫球蛋白的比较研究．中国水产科学，6(4)：109～110
11．杨桂文，安利国，王长法．鲤鱼皮肤粘液与血清中免疫球蛋白的比较研究．动物学研究，1998，19(6)：489～492
12．杨桂文，安利国，温武军等．鲤胆汁与血清中免疫球蛋白的比较研究．水产学报，1998，22(3)：199～203
13．陈昌福，纪国良，罗宇良等．草鱼体表和肠粘液中免疫球蛋白的初步分析．鱼病学研究论文集，1999，2：21～25
14．陈昌福．用直接荧光抗体法对鲤血液中淋巴细胞类型的初步鉴定．华中农业大学学报，1998，17(2)，154～158
15．周丽，战文斌，宋微波等．指状拟舟虫诱导牙鲆抗血清免疫球蛋白分析．中国水产科学，2000，7：28～31
16．孟凡国，李亚南．鱼类体液免疫研究进展．国外兽医学—畜禽疾病，1996，17(4)：16～18
17．林天龙，陈强，俞伏松等．欧洲鳗血清免疫球蛋白纯化及部分特性分析．水产学报，2000，25(1)：52～57
18．赵振山．沟鲶养殖．科学出版社，1994
19．钟明超，林浩然．硬骨鱼类胚胎和胚后发育阶段的免疫机制．中国水产科学，1996．3(2)：97～102
20．夏春，川合研儿，楠田理一．鳗鲡淋巴细胞表面存在不同表型的免疫球蛋白．水产学报，1996，20(4)：361～364
21．聂品．鱼类非特异性免疫研究的新进展．水产学报，1997，21(1)：69～73
22．郭尧君．蛋白质电泳实验技术．北京：科学出版社，1998
23．葛雷，常洪．斑点叉尾鮰小瓜虫病的防治．鱼类病害研究，1996，4：24
24．葛雷，常洪．斑点叉尾鮰 烂尾病的初步研究．鱼类病害研究，1996，4：23
25. Amemjya C T, Litmen G W. Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunogobulin gene organization in a primative teleost species. Proc Natl Acad Sci USA, 1990, 87: 811～815
26. Bang J D, Kim J W, Lee S D, et al. Humoral immune response of flounder to Edwardsiella tarda: the presence of various sizes of immunoglobulins in flounder. Dis Aqu Org, 1996, 26: 197～203
27. Bly J E, Grimm A S, Morris I G, et al. Transfer of passive immunity from mother to young in a teleost fish: Hemagglutinating activity in the serum and eggs of plaice, Pleuronectes platessa. L. Comp Biochem Pnysiol. 1986, 84A: 309～313
28. Bourmand C A F, Romestand B, Bouix G. Isolation and partial characterization of IgM-like seabass (Dicentrarchus labrax L.1758) immunoglobulins. Aquaculture, 1995, 132: 53～58
29. Bradshaw C M, Clem L M, Sigel M M. IgM antibodies in fish mucus. Proc Soc Exp Biol Med. 1971, 136: 1122
30. Chen changfu, Li Jing, Riichi Kusuda. Preliminary study on maternal immunity of grass carp (Ctenopharyngodon idellus). Journal of Huazhong Agricultural University. 1996,15(1),73～77
31. Clem L W et al. Evolution of lymphocyte subpopulations their interactions and temperature sensitivities. In: Warr G M, Cohen N. Ed. Phylogenesis of Immune Function. Boca Raton, Florida: CRC Press. 1991, 191～214
32. Clem L W. Phylogeny of immunoglobulin structure and function. Ⅳ. Immunoglobulins of the giant grouper, Epinephelus itaira, d Biol Chem Biological, 1971, 246: 9～15
33. Davidson G A et al. Route of immunization influences the generation of antibody secreting cells in the gut of rainbow trout (Oncorhynchus mykiss). Dev Comp Immunol, 1993, 17: 373～376
34. Davidson G A. Aspects of mucosal immunity in rainbow trout, Oncorhynchus mykiss(Walbaum 1792). PhD thesis, Aberdeen University. 1991.
35. Deluca D, Willson M, Warr G W et al. Lymphocyte heterogeneity in the trout, Salmo gairdneri, defined with monoclonal antibodies to IgM. Eur J Immunol, 1983, 13: 546～551
36. Dorson M. Applied immunology of fish. In: Symposium on fish vaccination (Ed:by De Kinkilin, P). 1984, 39～74.
37. Elcombe B M, Chang R J, Taves C J, et al. Evolution of antibody structure and effector functions: comparative hemolytic activities of monomeric and tetrameric IgM from rainbow trout, Salmo gairdneri. Comp Biochem Physiol, 80B: 697～706
38. Ellis E et al. Surface immunoglobulins on the lymphocytes of the skate Reja naevua. Eur J Immunol, 1975, 5: 726～728
39. Estevez J et al. Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays. Vet Immunol Immunopathol, 1994, 41: 353～366
40. Estevez J, Leiro J, Sanmartin M L, et al. Isolation and partial characterization of turbot (Scophthalmus maximus) immunoglobulins. Comp Biochem Physiol, 1993,1 05A: 275～281
41. Fletcher T C, Grant P T. Immunoglobulin in the serum and mucus of the plaice(Pleuronectes platessa),J Biochem, 1969, 115: 65
42. Fuda H, Hara Aj, Yamazaki F, et al. A speculiar immunoglobulin M (IgM) identified in eggs of chum salmo (Oncorhynchus keta). Dev Comp Immunol, 1992, 16: 415～423
43. Fuller L, Murray J, Jensen J A. Isolation from nurse shark serum or immune 7S antibodies with two different molecular weight H-chains. Immunochemistry, 1978, 15: 251～259
44. Ghaffari S H, Lobb C J. Cloning and sequence analysis of channel catfish heavy chain cDNA indicate phylogenetic diversity within the IgM immunoglobulin family. J Immunol, 1989, 142: 1356～1365
45. Ghaffari S H, Lobb C J. Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogenety of Ig heavy chains, J Immunology, 1989, 143: 2730～2739
46. Graham S, Secombes C J. Cellular requirements for lymphocykine secretion by rainbow trout Salmo gairdneri leucocytes. Dev Comp Immunol, 1990, 14: 59～68
47. Hagiwara k, Kobayashi K, Kajii T, et al. J-chain-like component in 18-S immunoglobulin of the skate Raja Kenojei. a cartilaginous fish. Mol Immunol, 1985, 22(7): 775～778
48. Harding F A, Cohen N, Litman G W. Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea. Nucleic Acids Res, 1990, 18(4): 1015～1020
49. Harding F A,Amemiya C T. Two distinct immunolobulin heavy chain isotypes in a primative, cartilaginous fish, Raja erinacea. Nucleic Acids Res, 1990, 18(21): 6369～6376.
50. Harlow, David Lane. Antibodies-A laboratory manual. Cold Spring Harbor Laboratory Press, NY, 1988
51. Harrell L W, Etlinger L M, Hodgins H O. Humoral factors important in resistance of salmonid fish to bacterial disease. Ⅱ. Anti-Vibro anguillarum activity in mucus and observations on complement. Aquaculture, 1976, 7: 363～370
52. Hart S, Warthmell A B, Harris J E, et al. Gut iummunology in fish: a review. Dev Comp Immunol, 1988, 12: 453～480
53. Havarstein L S, Assjord P M, Ness S, et al. Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar). Dev Comp Immunol, 1988, 12: 773～785
54. Hayman J R, Lobb C J. Immunoglobulin in the eggs of the channel catfish (Ictalurus punctatus). Dev Comp Immunol, 1993, 17: 241～248
55. Hung H W, Lo C T, Tseng C C, et al. Humoral immune response of Japanese eel, Anguilla japonica, to Pleistophora anguillarum Hoshina 1951(Microspora). J Fish Dis, 1996, 19: 243～250
56. Itami T et al. Purification and characterization of immunoglobulin in skin mucus and serum of ayu. Nippon Suisan Goakkaish, 1988, 54: 1611～1617
57. Jurd R D. Specialization in the teleost and anuran immune response. In: M J Manning, M F Tatner. A comparative critique in fish immunology. London: Academic Press. Inc, 1985, 9～28
58. Kobayashi K, Tomonaga S, Kajii T. A second class of immunoglobulin other than IgM present in the serum of a cartilaginous fish, the skate, Raja Kenjei: isolation and characterization. Mol Immunol, 1984, 21: 397～404.
59. Kobuku G, Litman R, Shambiott M J, et al. Diverse organization of immunoglobulin VH gene local in a Primitive vertebrate. EMBO, 1988, 7: 3413～3422
60. Laemmli U K. Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature(London), 1970, 227: 680～685
61. Lobb C J, Clem L W. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev Comp Immunol, 1982, 6: 473～479
62. Lobb C J, Clem L W. Immunoglobulin light chain classes in a teleost fish. J Immunol, 1984, 132: 1917～1923
63. Lobb C J, Clem L W. Phylogeny of immunoglobulin structure and function.X.Humoral immunoglobulins of the sheepshead, Archosargus probatocephalus. Dev Comp Immunol, 1981c, 5: 271～282
64. Lobb C J, Clem L W. The metabolic relationships of the immunoglobulins in fish serum, cutaneous mucus, and bile. J Immunol. 1981,127:1525
65. Lobb C J, Olson M O J. Immunoglobulin heavy H chain isotypes in a teleost fish. J Immunol, 1988, 141: 1236～1245
66. Lobb C J. Secretary immunity induced in catfish, Ictalurus punctatus, following bath immunization. Dev Comp Immunol, 1987, 11: 727～738
67. Lobb C J. Structural diversity of channel catfish immunoglobulins. Veterinary Immunology and Immunopathology, 1986, 12: 7～12
68. Loghothetic P N, Austin B, Immune response of rainbow trout (Oncorhynchus mykiss, Walbaum) to Aeromonas hydrophila. Fish Shellfish Immunol. 1994, 4: 239～254
69. Lumsden J S et al. Detection of a distinct gill-surface antibody response following horizontal infection and bath challenge of brook trout Salvelinus fontinalis with Flavobacterium branchiophilum, the causative agent of bacterial gill disease. Dis Aqua Org, 1993, 16: 21～27
70. Manning M J. Fishes. In: Tumer R J ed. Immunology comparative approach. Britain: John Wiley & Sons Ltd. 1994, 69～100
71. Mccumber L J, Clem L W. A comparative study of J chain: structure and stoichicometry in human and nurse shark IgM. Immunochemistry, 1976, 13, 479～484
72. McGhee J R, Mestecky J, Derzbaugh M J, et al. The mucosal immune system from fundamental concepts to vaccine development. Vaccine, 1992 10: 75～88
73. Mestecky J, Kulhavy R, Schrohenloher R E, et al. Identification and properties of J chain isolated from catfish macroglobulin. J Immunol, 1975, 115: 993～997
74. Mor A, Aviation R R. Evidence of transfer of immunity from mother to eggs in tilapias. Israeli J. Aquacult(Bamidgeh), 1988, 40: 22～28
75. Mor A, Avtalion R R. Transfer of antibody activity from immunized mother to embryo in tilapias, J Fish Biol. 1990, 37: 249～255
76. Nakao M, Moritomo T, Tomana M, et al. Isolation of cDNA encoding the constant region of the immunoglobulin heavy-chain from common carp. (Cyprinus carpio L.). Fish Shellfish Immunol, 1998, 8(6):425～434
77. Navarro V et al. Immuno(cyto)chemical characterization of monoclonal antibodies to githead seabream (Sparus aurata) immunoglobulin. Fish Shellfish Immunol, 1993, 3: 167～177
78. Nossal G J V, Sberg A A, GL,Austin C M. Single cell studies on 19s antibody production. J. Exp Med, 1964, 119: 485～502
79. Ourth D D. Purification and quantification of channel catfish (Ictalurus punctatus) immunoglobulin M. douranl of Applied Ichthyology, 1986, 3: 140～143
80. Phillips J O, Ourth D D. Isolation and molecular weight determination of two immunoglobulin heavy chains in the channel catfish, Ictalurus punctatus. Comparative Biochemistry and Physiology, 1986, 85B: 49～54
81. Pilstrom L, Peterson A. Isolation and partial characterization of immunoglobulin from aye (Gadus morhua L.). Dev Comp Immunol, 1991, 15: 143～152
82. Rabbits T H, Forster A, Dunmik W, Bentley D L. The role of gene deletion in the immunglobulin heavy chain switch. Nature, 1980, 283: 351～356.
83. Rombout J H W M, et al. Differences in mucus and serum immunoglobulin of carp (Cyprinius carpio L). Dev Comp Immunol, 1993, 17: 309～317
84. Rombout J H W M, Bot H E, Lamers C H J, et al. Immunological importance of the second gut segment of carp. Ⅱ. Characterization of mucosal leucocytes. J Fish Biol, 1989a, 35: 167～178
85. Rombout J H W M, Van den Berg A A, Van den Berg C T GS,et al. Immunological importance of the second gut segment of carp. Ⅱ. System and/or mucosal immune responses after immunization with soluble or particulate antigens. J Fish Biol, 1989b, 35: 179～186
86. Rombout J W H M, Blok L J, Lamers C H J, et al. Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system. Dev Comp Immunol, 1986, 10: 341～351
87. Sanchez C, Domiguez J, Coll J. Immunoglobulin heterogeneity in the rainbow trout, Salmo gairdneri Richardson. J Fish Dis, 1989, 12: 459～465
88. Sanchez C, Dominguez J. Trout immunoglobulin populations differing in light chains revealed by monoclonal antibodies. Mol Immunol, 1991, 28: 1271～1277
89. Sanchez C, Lopez-Fierro P, et al. Characterisation of monoclonal antibodies against heavy and light chains of trout immunoglobulin. Fish Shellfish Immunology, 1993, 3(4): 237～251
90. Scapigliati G, Romano N, et al. Monoclonal antibodies against sea bass Dicentrarchus labrax L. Immunoglobulins: immunolocalisation of immunoglobulin-bearing cells and applicability in immunoassays. Fish Shellfish Immunology, 1996,6(5):383～401
91. Secombes C J et al. The development of an ELISPOT assay to quantify total and specific antibody-secreting cells in dab Limanda limanda L. Fish Shellfish Immunol, 1991, 1: 87～97
92. Secompes C J et al. Separation of lymphocyte subpopulation in carp (Cyprinus carpio L) by monoclonal antibodies immunohistochemical studies. Immunol,1983, 48: 165～175
93. Service R F. Triggering the first line of defense. Science, 265: 1522～1524
94. Shelton E, Smith M. The ultrastucture of carp (Cyprinus carpio) immunoglobulin: a tetrameric Immunogllbulin. J. Molec. Biol. 1970, 54, 615～617
95. Smith S A, Gebhard D H, Housman J M et al. Isolation, purification and molecular-weight determination of serum immunoglobulin from Oreochromis aureus. Journal of Aquatic Animal Health, 1993, 5: 23～35
96. St Louis-Cormier E A, Osterland C K, Aderson P D et al. Evidence for a cutaneous secretory immune system in the rainbow trout (Salmo gairdneri). Dev Comp Immunol, 1984, 8:71
97. Takahashi Y, Kawahara E. Maternal immunity in newborn fry of the ovoviparous guppy. Nippon Suisan Gakkaishi, 1987,53:721～735
98. Takemura A. Changes in an immunoglobulin M(IgM)-like protein during larval stages in tilapia, Oreochromis mossambicus. Aquaculture, 1993,115:233～241
99. Thuvander A,Fossum C, Lorenzen N. Monocolnal antibodies to salmonid immunoglobulin: characterization and applicability to immunoassays. Dev Comp Immunol. 1990,14:415～423
100. Tomanaga S et al. Characterization of serum immunoglobulins in a chendrostean fish. Dev Comp Immunol.1993, 17: 515～524
101. Trump G N et al. Antibody response of goldfish to bovine serum albumin: Primary and secondary responses. Immunology, 1970,19:627
102. Tucker C S, Robinson E H. Channel catfish farming handbook. Elsevier Science Publishers. 1991.
103. Warr G W et al. Phylogenetic origins of immune recognition: lymphocyte surface immunoglobulin of the goldfish, carassius auratus. Proc Nat Acad Sci. 1976, 73: 2476～2480
104. Whittington K J. Purification and partial characterization of serum immunoglobulin of the European perch (Perca fluviatilis L.). Fish Shellfish Immunol, 1993, 3: 331-343
105. Wilson M R, Warr G W. Fish immunoglobulins and the genes that encode them. Annual Review of Fish Diseses. 1992, 2: 201～221
106. Wolf K, Quimby M C, Bradford A D, et al. Egg-associated transmission of IPN virus of trout. Virology, 1963, 21: 317～321
107. Xia C et al. Presence of mitogen receptors on the lymphocyte of eel, Anguilla japonica. Suisanzoshoku, 1994b, 42: 53～56
108. Xia C et al. Receptors for sheep red blood cells, imunoglobulin M and complement detected on the lymphocyte of eel, Anguilla japonica. Suisanzoshoku, 1994a, 42: 47～51
109. Xia C et al. Studies on the heterogeneity of lymphocyte population in eel, Anguilla japonica. Suisanzoshoku, 1993, 41: 119～123