摘要
噬菌体抗体库技术是近年来抗体技术研究中革命性的进展,利用此技术能够快速有效地制备人源化抗体,并且可以筛选到具有高亲和力的特异性抗体。目前此项技术在水产养殖业中尚未见报道,基于斑点叉尾鮰抗体基因研究比较清楚,本文构建了其天然噬菌体抗体库,具体工作如下:
1引物设计
斑点叉尾鮰重链可变区分为13个VH家族,针对每个VH家族设计1对特异性引物,共13对。轻链有两种类型,F链和G链,由于轻链基因序列同源性很高,因此每种类型设计1对引物,共2对。所有引物两端都带有限制性酶切位点,5'引物位于免疫球蛋白可变区前导肽和N端第一骨架区(FR1),3'位于CH1或CL末端。
2轻链库的构建
取50尾健康斑点叉尾鮰新鲜外周血共250mL,分离淋巴细胞,提取总RNA,经逆转录获得cDNA。PCR扩增抗体重链Fd和轻链基因片段,回收约650bp的抗体片段。将回收的PCR产物连接T载体(pT-Fd,pT-L),分别用SacI+XbaI,SpeI+XhoI酶切,再次回收重链Fd和轻链基因片段。将第二次回收的轻链基因片段插入噬菌粒载体p3MH,重组后电转化大肠杆菌XL1-BLue,构建轻链库p3MH-L。取适量电转化后的菌液铺板培养,统计转化子数,计算库容。经过多次转化轻链库库容达到5.10×10~7。随机挑取20个菌落,菌液PCR,电泳显示18个菌落可见约650bp的轻链基因条带,轻链库重组率为90%。将质粒p3MH-L经过SacI+XbaI酶切可见约650bp的插入片段,说明轻链库构建成功。
3抗体库的构建
将轻链库质粒p3MH-L经SpeI+XhoI双酶切,回收载体大片段(约4.1kb),将其与第二次回收的重链Fd段基因连接,转化大肠杆菌,按构建轻链库的方法计算转化率和插入效率。经过4次转化最终得到总库容为5.67×10~7的抗体库(p3MH-L-Fd),插入效率为90%。抗体库质粒经XhoI单酶切,SpeI+XhoI双酶切后可见插入的目的片段,说明抗体库构建成功。
4抗体库多样性分析
将构建的抗体库菌液铺平皿后随机挑取单菌落,摇菌后测定重链Fd和轻链基因序列,进行序列对比,分析抗体库多样性。共测定10条重链Fd段基因序列和6条轻链基因序列,结果显示扩增的Fd段和轻链基因结构完整,抗体多样性良好。
The antibody engineering technology has a revolutionary advance by using phage antibody libraries. It provides a powerful method to produce humanizd monoclonal antibodies and could obtain antibodies with higher affinity. The Fab phage antibody libraries of Channel catfish were constructed, because the biochemical information on structure and function of channel catfish immunogloblins had been known and there were no reports on the phage antibody library in aquaculture. The experiment results as following:
1 Primer design
In the catfish VH gene segments are organized into 13 different VH families for which we designed a pair of primers. Channel catfish has two classes of light chain (designated F and G) which have a high degree of sequence similarity, so we designed two pairs of primers. There were restriction enzymes sites on both sides of these primers. The 5'-primers were corresponding to regions within the leader sequence or N-terminal FR region and 3'-primers were corresponding to the CH1 or CL regions extremity.
2 Construction of the light chain library
Total RNA was extracted from the peripheral blood mononuclear cells (PBMCs) of 50 healthy channel catfish and then used to synthesize first strand cDNA. Heavy chain Fd fragments and light chain genes were amplified by a group of specific primers. The amplified products were purified and cloned into pMD18-T simple vector to get the recombinant vector pT-Fd, pT-L which were degested with restriction enzymes of SacI/XbaI or SpeI/XhoI and purified again in order to get heavy chain Fd and light chain genes. The second purifed products were inserted into the phagemid p3MH and then electro-transformed into competent E.coli XL1-Blue cells to construct the light chain library p3MH-L. The library members could be accounted by clones in agar medium flat. After several times of electroporations, the capacity of the light chain antibody library was 5.10×10~7. We selected 20 single colonies randomly which were amplified with primers of light chain and the bacilli PCR products showed that 18 fragments estimated to be 650bp in length were excised. The cloning efficiencies of light chain library were 90%. After degested with restriction enzymes of SacI/XbaI, the plasmid p3MH-L can be seen the insert size of about 650bp fragment which meaned that the light chain library was successfully constructed.
3 Construction of the phage antibody library
The phagemid vector p3MH-L was incised by endonucleases SpeI/XhoI and big segments (about 4.1kb) were collecd. The big segments and the second purifed heavy chain Fd fragments were conjugated by T4 DNA ligase and electro-transformed into competent E.coli XL1-Blue cells to construct Fab phage antibody library. We calculated its capacity and recombine rate just following the way of light chain library. After 4 times of electroporations, the results showed that the Fab phage antibody library contained 5.67×10~7 different clones and the recombine rate was 90%. The cutting of enzymes showed that there were heavy chain Fd fragment and light chain genes in the phagemid and we have successfully constructed a channel catfish na(?)ve Fab phage antibody library.
4 Analyzing the diversity of the antibody library
After construction of Fab phage antibody library, single colonies were randomly selected in order to determin heavy chain Fd fragment and light chain genes DNA sequences. Multiple sequence alignments indicated that heavy chain Fd fragment and light chain gene structure was entire, antibody diversity was fine.
引文
1.陈志南,刘民培.抗体分子与肿瘤.人民军医出版社,2003.
2.J.萨姆布鲁克,E.F.弗里奇,T曼尼阿迪斯著.金冬燕,黎孟枫等译.分子克隆实验指南第二版.北京:科学出版社,1993:917
3.王刚,刘玉峰,王琰,化冰.噬菌体抗体库几种筛选方法的比较.第四军医大学学报,2001,16:1482-1484
4.张春莉,王妍.抗体库技术.微生物免疫学进展,2001,29(4):86-89
5.Anupam K,Dattamajumdar,David P,Jacobson,Leroy E,Hood,Gamal E,Osman.Rapid cloning of any rearranged mouse immunoglobulin variable genes,Immunogenetics,1996,43:141-151
6.Barbas C F,Lerner R A.Combinatorial immunoglobulin libraries on the surface of phage(phabs):rapid selection of antigen specific Fabs.methods:A Companion to Methods in Enzymology.Neuroscience,1991,2:119-124.
7.Barbas C F,Burton D R,Scott J K,Silverman G J.Phage display:a laboratory manual.New York:Cold Spring Harbor Laboratory Press,2001
8.Barker K,Khayat M,Miller N,Wilson M,Clem L W,Bengten E.Immortal and mortal clonal lymphocyte lines from channel catfish:comparison of telomere length,telomerase activity,tumor suppressor and heat shock protein expression.Dev Comp Immunol,2002,26(1):45-51
9.Barker K S,Quiniou S M,Wilson M R,Bengten E,Stuge T B,Warr G W,Clem L W,Miller N W.Telomerase expression and telomere length in immortal leukocyte lines from channel catfish.Dev Comp Immunol,2000,24(6-7):583-95.
10.Benhar I,Nahary L,Shaky S.DIP selection of phage-antibodies mediated by antigen expression on live bacteria.Mol Biol,2000,301:893-904.
11.Bengten E,Str(o|¨)mberg S,Pilstr(o|¨)m L.Immunoglobulin VH regions in Atlantic cod(Gadus morhua L):their diversity and relationship to VH families from other species.Dev Comp Immunol,1994,18:109-122
12.Bengten E,Clem L W,Miller N W,Warr G W,Wilson M.Channel catfish immunoglobulins:repertoire and expression.Dev Comp Immunol,2006,30:77-92
13.Bly J E,Miller N W,Clem L W.A monoclonal antibody specific for neutrophils in normal and stressed channel catfish.Dev Comp Immunol,1990,14(2):211-221.
14.Bradbury A,Persic L,Werge T,Cattaneo A.Use of living colunms to select specific phage antibodies.Biotechnology,1993,11:1565-1569.
15. Bradbury A, Sblattero D. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnology, 2000, 18: 75-80
16. Brodeur P H, Osman G E, Mackle J J, Lalor T M. The organization of the mouse IgH-V locus: dispersion, interspersion, and the evolution of VH gene family clusters. Exp Med, 1988, 168:2261-2278
17. Burton D R, Barbas C F 3rd, Persson M A, Koeniq S, Chanock R M. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci USA, 1991, 88: 10134-10137
18. Caldas C, Coelho V, Kalil J, Moro A M, Maranhao A Q, Brigido M M. Humanization of the anti-CD 18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen. Mol Immnunol, 2003, 39(15): 941-952
19. Cai X, Garen A. Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc Natl Acad Sci USA, 1995, 92(14): 6537-6541
20. Chester K A, Begent R H, Robson L, Keep P, Pedley R B, Boden J A, Boxer G, Green A, Winter G, Cochet O. Phage libraries for generation of clinically useful antibodies. Lancet, 1994, 343:455-456
21. Clackson T, Hoogenboom H R, Griffiths A D, Winter G. Making antibody fragments using phage display libraries. Nature, 1991, 352(6336): 624-628
22. Davies J M, O'hehir R E, Suphioglu C. Use of phage display technology to investigate allergen-antibody interactions. Allergy Clin Immunol, 2000, 105: 1085-1092
23. Desai S A, Wang X, Noronha E J, Kageshita T, Soldano F. Characterization of human anti-high molecular weigh-melanoma-associated antigen single-chain Fv fragments isolated from a phage display antibody library. Cancer Research, 1998, 58(11): 2417-2425.
24. Foigori A, Tafi R, Meola C. A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO J, 1994, 13(9): 2236-2243
25. Getahun A, Lundqvist M, Middleton D, Warr G, Pilstrom L. Influence of the mu-chain C-terminal sequence on polymerization of immunoglobulin M. Immunology, 1999, 97(3):408-413
26. Ghaffari S H, Lobb C J. Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains. Immunol, 1989, 143(8):2730-2739
27. Ghaffari S H, Lobb C J. Heavy chain variable region gene families evolved early in phylogeny Ig complexity in fish. Immunol, 1991, 146(3): 1037-1046
28. Ghaffari S H, Lobb C J. Structure and genomic organization of immuboglobulin light chain in the channel catfish.An umusual genomic organizational pattern of segmental genes. Immunol, 1993,151:6900-6912
29. Griffiths A D, Malmqvist M, Marks J D, Bye J M, Embleton M J, Baier M, Holliqer K P, Gorick B D, Huqhes-Jones N C. Human anti-self antibodies with high specificity from phage display libraries. EMBO J, 1993, 12: 725-734
30. Griffiths A R, Duncan A R. Strategies for selection of antibodies by phage display. Curr Opin Biotechnol, 1998, 9: 102-108
31. Hayman J R, Ghaffari S H, Lobb C J. Heavy chain joining region segments of the channel catfish.genomic organization and phylogenetic implications. Immunol, 1993, 151: 3587-3596
32. Hayman J R, Lobb C J. Heavy chain diversity region segments of the channel catfish: structure,organization, expression and phylogenetic implications. Immunol, 2000, 164: 1916-1924
33. Hinds-Frey K R, Nishikata H, Litman R T, Litman G W. Somatic variation precedes extensive diversification of germline sequences and combinatorial joining in the evolution of immunoglobulin heavy chain diversity. Exp Med, 1993, 178: 815-824
34. Hsu E, Steiner L A. Primary structure of immunoglobulin through evolution. Curr Opin Struct Biol, 1992,2:422-431
35. Ishida T, Tmai K. The expression technology of chimeric and humanized antibodies. Nippon Rinsho, 2002, 60(3): 439-444
36. Johns M., George A J, Ritter M A. In vivo selection of scFv from phage display libraries.Immunol Methods, 2000,239: 137-151
37. Kabat E A, Wu T T. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. Immunol 1991, 147:1709-1728
38. Kabat E A, Wu T T, Perry H M, Gottesman K S, Foeller C. Sequences of proteins of immunological Interest. National Institutes of Health, Bethesda, MD, 1991
39. Khayat M, Stuge T B, Wilson M, Bengten E, Miller N W, Clem L W. Thioredoxin acts as aB cell growth factor in channel catfish. Immunol, 2001, 166(5): 2937-2943
40. Knight K L. Restricted VH usage and generation of antibody diversity in rabbit. Ann Rev Immunol, 1992, 10: 593-616
41. Kohler G, Milstein C. Continuous cultures of fused cells secretion antibody of predefined specificity. Nature, 1975, 256: 495-497
42. Kokubu F, Litman R, Shamblott M J, Hinds K. Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate. EMBO J, 1988, 7: 3413-3422
43. Ledford B E, Magor B G, Middleton D L, Miller R L, Wilson M R, Miller N W, Clem L W, Warr G W. Expression of a mousechannel catfish chimeric IgM molecule in a mouse myeloma cell.Mol Immunol, 1993,30(16): 1405-1417
44. Li Y, Cockburn W, Kilpatrick J B, Whitelam G C. High affinity ScFvs from a single rabbit immunized with multiple haptens. Biochemical and Biophysical Research Communication, 2000,268: 398-404
45. Lin G L, Ellsaesser C F, Clem L W, Miller N W. Phorbol ester/calcium ionophore activate fish leukocytes and induce long-term cultures. Dev Comp Immunol, 1992, 16(2-3): 153-63
46. Lobb C J, Clem L W. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev Comp Immunol, 1982, 6(3): 473-479
47. Lobb C J, Clem L W. Distinctive subpopulations of catfish serum antibody and immunoglobulin.Mol Immunol, 1983,20: 811-818
48. Lobb C J, Olson M O, Clem L W. Immunoglobulin light chain classes in a teleost fish. Immunol,1984, 132(4): 1917-23.
49. Lobb C J. Covalent structure and affinity of channel catfish anti-dinitrophenyl antibodies. Mol Immunol, 1985, 22(8): 993-999
50. Lobb C J. Structural diversity of channel catfish immunoglobulins. Vet Immunol Immunopathol,1986, 12(1-4): 7-12.
51. Lobb C J. Preferential expression of catfish light chain immunoglobulin isotypes in anti-dinitrophenyl antibodies. Immunogenet, 1986, 13(1): 19—28
52. Lundqvist M L, Pilstrom L. Variability of the immunoglobulin light chain in the Siberian sturgeon, Acipenser baeri. Dev Comp Immunol, 1999, 23: 607-615
53. Liu B, Marks J D. Applying phage antibodies to proteomics: selecting single chain Fv antibodies to Antigens blotted on nitrocelluglose. Analytical Biochem, 2000, 286: 119-128
54. Magor B G, Ross D A, Pilstrom L, Warr G W. Transcriptional enhancers and the evolution of the IgH locus. Immunol Today, 1999, 20: 13-17
55. Mao C, Flynn C E, Hayhurst A, Sweeney R, Qi J, Georgiou G, Iverson B, Belcher A M. Viral assembly of oriented quantum dot nanowires. Proc Natl Acad Sci USA, 2003, 100(12): 6946-6951
56. Marks JD, Tristem M, KarPas A, Winter G. Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes. Eur J Immunol, 1991, 21: 985-991
57. Matsuda F, Ishii K, Bourvagnet P, Kuma K, Hayashida H, Miyata T, Honjo T. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. Exp Med,1998,188:2151-2162
58. Max, E E, Immunoglobulins: molecular genetics. In: Paul, W.E.E.D., Fundamental Immunology.Lippincott-Raven, Philadelphia, 1999, 111-182
59. McCormack W T, Carlson L M, Tjoelker L W. Evolutionary comparison of the avian IgL locus: combinatorial diversity plays a role in the generation of the antibody repertoire in some avian specie. Int Immunol, 1989, 1: 332-341
60. Miller N W, Sizemore R C, Clem L W. Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro antibody responses of channel catfish leukocytes. Immunol, 1985, 134(5):2884-2888
61. Miller N W, Bly J E, van Ginkel F, Ellsaesser C F, Clem L W. Phylogeny of lymphocyte heterogeneity: identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies. Dev Comp Immunol, 1987, 11(4): 739-47
62. Miller N W, Rycyzyn M A, Wilson M R, Warr G W, Naftel J P, Clem L W. Development and characterization of channel catfish long term B cell lines. Immunol, 1994, 152(5): 2180-2189
63. Miller S A, Dykes D D, Polesky H F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 1998, 16: 1215-1219
64. Ota T, Sitnikova T, Nei M. Evolution of vertebrate immunoglobulin variable gene segments. Curr Top Microbiol Immunol, 2000, (248): 221-245
65. Pasquier L D, Wilson M, Greenberg A S, Flajnik M F. Somatic mutation in ectothermic vertebrates: musing on selection and origins. Curr Top Microbilo Immunol, 1998, 229: 199-216
66. Pereira S, Van Belle P, Elder D, Maruyama H, Jacob L, Sivanandham M, Wallack M, Sieqel D,Herlyn D. Combinatorial antibodies against human malignant melanoma. Hybridoma, 1997, 16:11-17
67. Pilstrom L, Bengten E. Immunoglobulin in fish-genes, expression and structure. Fish Shellfish Immunol, 1996, 6: 243-262
68. Rodriguez-Diaz J, Monedero V, Perez-Martinez G, Buesa J. Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display. J Virol Methods, 2004,121(2): 231-235
69. Roman T, De Guerra A, Charlemagne J. Evolution of specific antigen recognition: size reduction and restricted length distribution of the CDRH3 regions in the rainbow trout. Eur J Immunol,1995, 25: 269-273
70. Roman T, Andersson E, Bengten E. Unified nomenclature of Ig VH genes in rainbow trout (Oncorhynchus mykiss): definition of eleven VH families. Immunogenetics, 1996, 43: 325-326
71. Sblattero D, Bradbury A A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology, 1998, 3: 271-278
72. Sharon J, Gefter M L, Manser T, Morrison SL, Oi V T, Ptashne M. Expression of a V H C kappa chimaeric protein in mouse myeloma cells. Nature, 1984, 309(24): 364-367
73. Shen L, Stuge T B, Zhou H, Khayat M, Barker K S, Quiniou S M, Wilson M, Bengten E,Chinchar G, Clem L W, Miller NW. Channel catfish cytotoxic cells: a mini-review. Dev Comp Immunol, 2002, 26(2): 141-149
74. Shen L, Stuge T B, Evenhuis J P, Bengten E, Wilson M, Chinchar V G, Channel catfish NK-like cells are armed with IgM via a putative FcuR. Dev Comp Immunol, 2003, 699-714
75. Siegel D L, Chang T Y, Russell S L, Bunyav Y. Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting : applications in immunohematology. Immunol Methods, 1997, 206: 73-85
76. Skerra A, Pl(?)ckthurn A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science, 1988, 240 (4855): 1038-1079.
77. S(?)derfind E, Lagerkvist A C, Duenas M, Malmborg A C, Ayala M, Danielsson L, Borrebaeck K. Chaperonin assisted phage display of antibody fragments on filamentous bacterio phages.Biotechnology, 1993, 11: 503-507
78. Sodoyer R, Peubez I, Pion C, Dubayle J, Jacquemot P, Geoffroy F, Even J, Aujame L. Full-scale naive human antibody repertoires assembled from VH and VL variable regions. Hum Antibodies,1997,8:37-42
79. Srisapoome P, Ohira T, Hirono I, Aoki T. Genes of the constant regions of functional immunoglobulin heavy chain of Japanese flounder Paralichthys olivaceus. Immunogenetics, 2004,56 (4): 292-300.
80. Stenvik J, Lundback A S, J(?)rgensen T Q. Variable region diversity of the Atlantic cod (Godua morhua L) immunoglobulin heavy chain. Immunogenetics, 2000, 51: 670-680
81. Stuge T B, Yoshida S H, Chinchar V G, Miller N W, Clem L W. Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures. Cell Immunol,1997, 177(2): 154-161
82. Stage T B, Wilson M R, Zhou H, Barker K S, Bengten E, Chinchar G, Miller N W, Clem L W.Development and analysis of various clonal alloantigen-dependent cytotoxic cell lines from channel catfish. Immunol, 2000, 164(6): 2971-2977
83. Tonegawa S. Somatic diversification of antibody diversity. Nature, 1983, 302-575
84. Tsurushita N, Fu H, Warren C. Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries. Gene, 1996,172: 59-63
85. Tutter A, Brodeur P, Shlomchik M, Riblet R. Structure, map position, and evolution of two newly diverged mouse Ig VH gene families. Immunol, 1991, 147: 3215-3223
86. Vallejo A N, Ellsaesser C F, Miller N W, Clem L W. Spontaneous development of functionally active long-term monocytelike cell lines from channel catfish. In Vitro Cell Dev Biol, 1991, 27(4):279-86
87. van Ginkel F W, Miller N W, Lobb C J, Clem L W. Characterization of anti-hapten antibodies generated in vitro by channel catfish peripheral blood lymphocytes. Dev Comp Immunol, 1992,16(2-3): 139-51
88. Ventura-Holman T, Jones J C, Ghaffari S H, Lobb C J. Structure and genomic organization of VH gene segments in the channel catfish: members of different VH gene families are interspersed and closely linked. Mol Immunol, 1994, 31: 823-832
89. Vaughan T J, Williams A J, Pritchard K. Human antibodies with sub-nanomolar affinities isolated from a large non-inununized phage display library. Nat Biotechnol, 1996, 14: 309-314
90. Ventura-Holman T, Ghaffari S H, Lobb C J. Characterization of a seventh family of immunoglobulin heavy chain VH gene segments in the Channel catfish, Ictalurus punctatus. EurJ Immunogenet, 1996, 23: 7-14
91. Ventura-Holman T, Lobb C J. Structural organization of the immunoglobulin heavy chain locus in the channel catfish: the IgH locus represents a composite of two gene clusters. Mol Immunol,2002, 38:557-564
92. Wilson M R, Marcuz A, van Ginkel F, Miller N W, Clem L W, Middleton D, Warr G W. The immunoglobulin M heavy chain constant region gene of the channel catfish, Ictalurus punctatus: an unusual mRNA splice pattern produces the membrane form of the molecule. Nucleic Acids Res,1990, 18(17): 5227-5233
93. Wilson M, Hsu E, Marcuz A, Courtet M, Pasquier L D, Steinberg C. What limits affinity maturation of antibodies in Xenopus-The rate of somatic mutation or the ability to select mutants?EMBOJ, 1992, 11: 4337-4347
94. Wu T T, Johnson G, Kabat E A. Length distribution of CDRH3 in antibodies. Proteins, 1993, 16:1-7
95. Xu B, Seising E. Analysis of sequence transfers resembling gene conversion in a mouse antibody transgene.Science, 1994,265: 1590-1593
96. Yancopoulos G D, Alt F W. Regulation of the assembly and expression of variable region genes,Annu Rev Immunol, 1986,4: 339
97. Yang F, Ventura-Holman T, Waldbieser G C, Lobb C J. Structure, genomic organization, and phylogenetic implications of six new VH families in the channel catfish. Mol Immunol, 2003, 40:247-260
98. Zhou H, Stuge T B, Miller N W, Bengten E, Naftel J P, Bernanke J M, Chinchar V G, Clem L W,Wilson M. Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed.Immunol, 2001, 167(3): 1325-1332
