鸡源抗禽流感病毒Fab噬菌体抗体库的构建研究
摘要
禽流感(avian influenza)是由A型流感病毒引起的一种禽类感染或疾病综合症。禽流感病毒属正粘病毒科(Orthomyxoviridae),为单股负链RNA病毒。甲型流感病毒除感染人外,还可感染猪、马、海洋哺乳动物和禽类。此病毒极易在禽鸟间散播,除可引起家禽大量死亡外,也可引起感染人的死亡。因此,禽流感病毒是一种危害禽类和人类健康的重要病原微生物。
对鸡源抗禽流感病毒抗体的研究不仅对疾病的预防和治疗有一定的作用,而且对进一步研究病毒在体内的致病机制提供了有利的工具。
我们首次运用噬菌体抗体库技术构建鸡源抗禽流感病毒Fab噬菌体抗体库。首先,我们从免疫鸡中提取外周血和脾脏,分离淋巴细胞,提取细胞总RNA,以其为模板,采用Oligo-dT引物逆转录合成cDNA,用一组鸡IgG Fab特异性引物,通过PCR方法扩增出一组抗体轻链基因和重链Fd片段基因。将抗体轻链基因克隆入pComb3噬菌粒载体,转化大肠杆菌XLI-Blue构建了库容量为2.5×10~5的轻链基因抗体库,轻链基因的克隆效率约为50%。进一步将抗体重链Fd基因克隆入轻链抗体库中,转化大肠杆菌,构建了库容量为1×10~5的鸡源抗禽流感病毒Fab抗体库。经鉴定,抗体轻、重链基因的克隆效率为40%。经辅助噬菌体感染,得到噬菌体滴度1×10~(13)CFU/ml的鸡源噬菌体抗体库。用固相化AIVAg,对所构建的噬菌体抗体库进行了五轮的富集,第五轮洗脱下的噬菌体较第一轮增加了100倍,含有抗体轻链基因和重链Fd基因的克隆,也由富集前的15%增至65%。挑取几个阳性克隆进行测序,获得的序列与GeneBank相应的鸡IgG基因序列具有较高的同源性。
鸡源抗禽流感病毒Fab噬菌体抗体库的构建成功为抗禽流感病毒特异性抗体的筛选及构建鸡源抗禽流感病毒全抗体库打下了良好的基础,并为进一步研制用于禽流感治疗和抗禽流感病毒单抗制剂奠定了基础,同时也为研究禽流感病毒的体内致病机制提供了条件。
Avian influenza (AI) is a disease infected by influenzavirus A. Avian influenza virus is the major etiologic agent of orthomyxoviridae. Influenzavirus A can infect swine, horse, mammal in ocean, poultry and human. Poultry and human which infected by this disease will also die.Phage display of antibody libraries has provided a powerful tool for the isolation of important viral pathogens .Large repertoires of antibodies can be displayed on the surface of filamentous phage particles, and antibodies with desired specificity can be isolated by panning on the antigen of interest.In this article ,we report the construction of chicken combinatorial immunoglobulin library to Avian Influenza Virus.The mRNA isolated from peripheral blood lymphocytes of chickens with AIV antibodies were reverse transcribed to the first strand cDNA using Oligo-dT as the primer.The genes of heavy Fd fragment and light chain of antibodies were amplified by a group of specific primers for chicken IgG Fab fragment. The gel purified light chain genes were firstly cloned into phagmid vector pComb3 to construct a chicken recombinatorial immunoglobulin library. The constructed library size was 1 x 10~5,and the cloning efficiencies of light chain genes were 50%.The heavy chain genes were subsequently combined with light chain genes randomly to constructed a combinatorial vectors.then transformed to E.Coli.XLI-Blue. the combinatorial Fabs libraries were constructed successfully. The chicken combinatorial Fab immunoglobulin library quantities were about 2.5x10~5 and the cloning efficiencies of Fab genes were 40%. Finally, the phage antibody library was further prepared by infecting chicken Fab combinatorial immunoglobulin library with help phage for displaying the titer of Fab fragment on the surface of phage .The titer of phages in library was lx10~13CFU/ml .The phage displaying antibody fragments were subjected to five rounds of paning with AIV antigen coated in solid phage. The eluted phages were enriched nearly 100-folder,and the percentase of recombinant clones increased from 15% to 65% after the five rounds of panning, choosed severy positive clones were sequenced and the data was analyzed by aligment with GeneBank, chicken immunoglobulin sequence was identified and confirmed. The light chains were identified as k type.We constructed chicken Fab recombinant library to AIV successfully. This specific Fab antibody combinatorial library laid a basis on panning and secreening specific Fab antibody to avian influenza virus. The antibodies panned and screened with puried AIV will be used as a tool to diagnose and study AIV. The antibodies will also be used as potential therapeutic medicine for the therapy of avian influenza after exposure to the virus.
对鸡源抗禽流感病毒抗体的研究不仅对疾病的预防和治疗有一定的作用,而且对进一步研究病毒在体内的致病机制提供了有利的工具。
我们首次运用噬菌体抗体库技术构建鸡源抗禽流感病毒Fab噬菌体抗体库。首先,我们从免疫鸡中提取外周血和脾脏,分离淋巴细胞,提取细胞总RNA,以其为模板,采用Oligo-dT引物逆转录合成cDNA,用一组鸡IgG Fab特异性引物,通过PCR方法扩增出一组抗体轻链基因和重链Fd片段基因。将抗体轻链基因克隆入pComb3噬菌粒载体,转化大肠杆菌XLI-Blue构建了库容量为2.5×10~5的轻链基因抗体库,轻链基因的克隆效率约为50%。进一步将抗体重链Fd基因克隆入轻链抗体库中,转化大肠杆菌,构建了库容量为1×10~5的鸡源抗禽流感病毒Fab抗体库。经鉴定,抗体轻、重链基因的克隆效率为40%。经辅助噬菌体感染,得到噬菌体滴度1×10~(13)CFU/ml的鸡源噬菌体抗体库。用固相化AIVAg,对所构建的噬菌体抗体库进行了五轮的富集,第五轮洗脱下的噬菌体较第一轮增加了100倍,含有抗体轻链基因和重链Fd基因的克隆,也由富集前的15%增至65%。挑取几个阳性克隆进行测序,获得的序列与GeneBank相应的鸡IgG基因序列具有较高的同源性。
鸡源抗禽流感病毒Fab噬菌体抗体库的构建成功为抗禽流感病毒特异性抗体的筛选及构建鸡源抗禽流感病毒全抗体库打下了良好的基础,并为进一步研制用于禽流感治疗和抗禽流感病毒单抗制剂奠定了基础,同时也为研究禽流感病毒的体内致病机制提供了条件。
Avian influenza (AI) is a disease infected by influenzavirus A. Avian influenza virus is the major etiologic agent of orthomyxoviridae. Influenzavirus A can infect swine, horse, mammal in ocean, poultry and human. Poultry and human which infected by this disease will also die.Phage display of antibody libraries has provided a powerful tool for the isolation of important viral pathogens .Large repertoires of antibodies can be displayed on the surface of filamentous phage particles, and antibodies with desired specificity can be isolated by panning on the antigen of interest.In this article ,we report the construction of chicken combinatorial immunoglobulin library to Avian Influenza Virus.The mRNA isolated from peripheral blood lymphocytes of chickens with AIV antibodies were reverse transcribed to the first strand cDNA using Oligo-dT as the primer.The genes of heavy Fd fragment and light chain of antibodies were amplified by a group of specific primers for chicken IgG Fab fragment. The gel purified light chain genes were firstly cloned into phagmid vector pComb3 to construct a chicken recombinatorial immunoglobulin library. The constructed library size was 1 x 10~5,and the cloning efficiencies of light chain genes were 50%.The heavy chain genes were subsequently combined with light chain genes randomly to constructed a combinatorial vectors.then transformed to E.Coli.XLI-Blue. the combinatorial Fabs libraries were constructed successfully. The chicken combinatorial Fab immunoglobulin library quantities were about 2.5x10~5 and the cloning efficiencies of Fab genes were 40%. Finally, the phage antibody library was further prepared by infecting chicken Fab combinatorial immunoglobulin library with help phage for displaying the titer of Fab fragment on the surface of phage .The titer of phages in library was lx10~13CFU/ml .The phage displaying antibody fragments were subjected to five rounds of paning with AIV antigen coated in solid phage. The eluted phages were enriched nearly 100-folder,and the percentase of recombinant clones increased from 15% to 65% after the five rounds of panning, choosed severy positive clones were sequenced and the data was analyzed by aligment with GeneBank, chicken immunoglobulin sequence was identified and confirmed. The light chains were identified as k type.We constructed chicken Fab recombinant library to AIV successfully. This specific Fab antibody combinatorial library laid a basis on panning and secreening specific Fab antibody to avian influenza virus. The antibodies panned and screened with puried AIV will be used as a tool to diagnose and study AIV. The antibodies will also be used as potential therapeutic medicine for the therapy of avian influenza after exposure to the virus.
引文
[1] 于康震,付朝阳,崔尚金,等.我国禽流感防制研究进展[J].中国兽医学报,2001,21(1):1-4.
[2] Kohler G, Mistein. Continuous cultures of fused cells secreting antibody predefined specificity[J]. Nature, 1975, 256:495-513.
[3] Li Haiyan, Xin Xiaoguang, Tian Guobin, et al. Dot-enzyme-linked immunosorbent assay for detection of antibodies to avian influenza virus [J]. Chin J Preventive veterinary medicine. 1999, 21(5):321—324.
[4] 李海燕,于震康,辛晓光,等.禽流感病毒重组核蛋白ELISA诊断技术的研究[J].中国预防兽医学报.2000,22(3):182—185.
[5] Li Haiyan, Yu Kangzhen, Xin Xiaoguang, et al. Development and validation of indirect enzyme-linked immunosorbent assay test kit for detecting anti-influenza virus antibodies [J]. Chin J Preventive Veterinary Medicine. 2001, 23(5):372—376.
[6] 唐秀英,田国斌,于震康,等.禽流感油乳剂灭活疫苗的研究.中国预防兽医学报[J].1999,21(6):401—405.
[7] 罗开健,辛朝安,薛义山,等.禽流感二价抗原油乳剂灭活疫苗研制[J].2000,30(7):3-5.
[8] Suarez DL, Schltz-Cherry S. Immunology of avian influenza virus: a review[J]. Developmental and comparative immunology[J]. 2000, 24:269-283.
[9] Winter G, Giffith AD, Hawkins RE, et al. Making antibodies by phage display technology [J] Annu Rev Immunol, 1994, 12:433-455.
[10] Relph Rapley. The biotechnology and applications of antibody engineering[J]. Molecul Biotechnol, 1995, 3:139-151.
[11] Hoogenboom HR, De Bruine AP, Hufton SE, et al. Antibody phage display technology and its applications [J]. Immunotechnology, 1998, 4:1-20.
[12] Lu J, Sloan SR, An alternating selection strategy for cloning phage display antibodies [J]. Immunol Methods, 1999, 228(1-2): 109-119.
[13] 万英,代佳平,王燕,等.一种稳定、易于操作的噬菌体随机肽库载体的构建[J].第三军医大学学报,1999,21(4):256-258.
[14] Skerra A, Pluckthum A, Assembly of a functional immunoglobulin Fv fragment in Escherichia Coli[J]. Science, 1988, 240:1038.
[15] Better M, Chang CP, Robinson RR, et al. Escherichia Coli secretion of an active chimeric antibody fragment [J]. Science, 1988, 240:1041.
[16] Barbas CF. Kang AS. Et al. Assembly of combinatorial antibody libraries on phage surface: The gene Ⅲ site [J]. Science. USA 1991, 88:7978.
[17] Hoogenboom HK, Griffiths AD. et al. Malti-subunit Proteins on the surface of of filamentous phage methodologies for displaying antibody(Fab) heavy and light chain [J]. Nucleic Acids Res. 1991, 19: 4133.
[18] Jesper LS, Messens JH, Keyser AD, et al. Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene Ⅵ [J]. Bio Technology,1995, 13: 378-382.
[19] Gap C, Map S, Lo CH, et al. Marking artificial antibodies: a format for phage display of combinatorial heterodimeric arrays [J]. Science, 1999, 96(11):6025-6030.
[20] Alting-Mess MA and JM. Polycos vectors:a system for packing filamentous phage and phagemid vectors using lambda phage packing extracts[J]. Gene, 1993, 137:93-100.
[21] Tsurshita N, Fu H and Warren C. Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries[J]. Gene, 1996, 172:59-63.
[22] Den W, Sompuram SR, Sarantopoulos S, et al. A bi-directional phage display vector for the selection and mass transfer of polyclonal antibody libraries[J]. Immunol, Methods 1999, 222:45-57.
[23] Hawkins RE, Russell SJ, Winter G, Selection of phage antibodies by binding affinity mimicking affinity maturation[J]. Mol Biol, 1992,4: 1-20.
[24] Hoogenboom HR, Bruine AP, Hufton SE, et al. Antibody phage display technology and its applications[J]. Immunotechnology, 1998, 4: 1-20.
[25] Tordsson J, Abrahmsen L, Kalland T, et al. Efficient selection of ScFv antibody phage by adsorption in situ expressed antigens in tissue sections[J]. Immunol Methods, 1997, 210: 11-23.
[26] Johns M, George JT, Ritter MA. In vivo selection of ScFv from phage display libraries[J]. Immunol Methods, 2000, 239:137-151.
[27] Osbourn JK, Derbyshire EJ, Vaughan TJ, et al. Pathfinder selection; in situ isolation of novel antibodies, Immunotechnology, 1998, 3: 293-302.
[28] Ward RL, Clark Ma, Lees J, et al. Retrieval of human antibodies from phage-display libraries using enzymatic cleavage[J]. Immunol Methods, 1996, 189: 73-82.
[29] Ridgway JBB, Ng E. Kern JA, et al. Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell line[J]. Cancer Res, 2000, 59:2718-2723.
[30] Ditzel HJ, Binley JM, Moore JP, et al. Neturalizing Recombinant human antibodies to a conformational V2-and CD4 binding site sensitive epitope of HIV-gp120 isolated by using an epitope-masking procedure[J]. Immunol, 1995, 154: 893-906.
[31] 王琰,刘群英,化冰,等.从人源性噬菌体抗体库分离出一株有异常序列的抗HBsAg Fab克隆.中国免疫学杂志[J].1998,14:115-118.
[32] Rojas G, A lmagro JC, Acevedo B, et al. Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable region [J]. J Biotechnol, 2002, 94 (3): 287—298.
[33] 王琰.抗体库技术[J].单克隆抗体通讯,1993,(9):23.
[34] Aujame L, Geoffroy F, Sodoyer. High affinity human antibodies by phage display. Human Antibodies. 1997, 8: 155-168.
[35] 胡建林,郭先健,胡义德,等.肺癌患者噬菌体抗体库的构建及抗肺癌单抗的筛选[J].免疫学杂志,2002,18(4):64-68.
[36] 何进,秦新裕,刘康达,等.胃癌相关的人源单链抗体基因文库的构建[J].实用癌症杂志,2002,17(2):113-115.
[37] Wang Zhiyi, Liu Qi, Wan Zesheng, et al. Construction of human combinatorial immunoglobulinli library containing HbsAb on the surface of phage[J]. Chin J Hepatol, 1999, (7): 159—161.
[38] 余宙耀,粟宽源,任向荣,等.工程化人源性抗HBs Fab抗体的制备、纯化和鉴定[J].上海免疫学杂志.1999,19(2):91—93.
[39] 尤长宣,罗荣城,丁雪梅.基因工程技术制备人源化抗HbsAg抗体Fab片段[J].上海免疫学杂志.2000,20(1):33—37.
[40] 王刚,王琰,化冰.基因工程抗角蛋白抗体的制备与鉴定.海军总医院学报[J].2002,15(1):9—13.
[41] Griffiths A D, Malmqvist M, Marks J D, et al. Human anti-self antibodies with high specificity from phage display libraries. [J]. EMBO J, 1993, 12(2):725-734.
[42] Marks J D, Hoogenboom H R, Bonnert T P, et al. By-passing immunization; human antibodies from V-gene libraries displayed on phage [J]. J Mol Biol, 1991, 222(3):581-597.
[43] de Haard H J, van Neer N, Reurs A, et, al. A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies[J]. J Biol Chem, 1999, 274(26): 18218-18230.
[44] 郭庆东,章翔,费舟,等.大库容量人源性天然单链抗体库的构建[J].第四军医大学学报,2001,22(23):2158-2161.
[45] Wang Gang, Wang Yan, Hua Bing, ea al. Construction of a large naive human antibody librayy [J]. Chin J Cell Mol Immunol, 2002, 18(1): 69-72.
[46] Bender E, Pilkington GR, Burton DR. Human monoclonal Fab Fragments from a combinatorial library prepared from an individual with a low serum titer to a virus [J]. H um A ntbod Hybridanas, 1994;5:2.
[47] Burton DR, Barbas CF Ⅲ, Persson MAA, et al. A large array of human an monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptom atic individual [J]. Proc Natl Acad Sci USA, 1991, 88:10134.
[48] Barbas CF Ⅲ, Crowe JE, Cababa D, et al Human an monoclonal Fab fraqments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein andneutralize infectivity [J]. Proc Natl Acad Sci USA, 1992, 89: 10164.
[49] Aujame L, Geoffroy F, Sodoyer. High affinity human antibodies by phage display[J]. Human Antibodies. 1997, 8: 155-168.
[50] Jespers LS, Roberts S, Mahler SM, et al. Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen[J]. Biotechnology-N-Y, 1994, 12:899.
[51] 刘志刚,俞炜源,林建波,等.人源化鼠抗人纤维蛋白单链抗体的体外成熟[J].细胞与分子免疫学杂志,2001,17(5):462-463.
[52] 程巨龙,王祥斌,张众,等.一种构建改形单域抗体的方法[J].遗传学报,2002,29(3):189-195.
[53] Sheets MD, Amersdorfer P, Finnern R, et al. Efficient construction of a large nonimmune phage antibody library: the production of high affinity human single-chain antibodies to protein antigens[J]. Proc Natl Acad Sci USA, 1998, 95:6157.
[54] Turner DJ, Ritter MA, George AJT, et al. Importance of the linker in expression of single chain Fv antibody fragments: optimization of pepitide sequence using phage display technology[J]. Immunol Methods, 1997,(205):43-54.
[55] Hennecke F, Krebber C, Pluckthum A, et al. Non-repetitive single-chain Fv linkers selectively infective phage(SIP)technology[J]. Protein Engng, 1998,(11):405-410.
[56] 陈晓穗,朱迎春,王欲晓,等.抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达[J].中国免疫学杂志,2000,16(5):248-25.
[57] Coia G, Ayres A, Lilley GG, et al. Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against hepatitis B[J]. Gene, 1997, 201: 203-209.
[58] Saviranta P, Pajunen M, Jauria P, et al. Engineering the steroid-specificity of an anti-17β-estradiol Fab by radom mutagenesis and competitive phage panning[J]. Protein Engng, 1998, 11: 143-152.
[59] Iba Y, Hayashi N, Sawada J, et al. Change in the specificity of antibodies against steroid antigens by introduction of mutation into complementarity-dtermining regions of the V_H domin. [J] Protein Engng, 1998, 11: 361-370.
[60] Tsui P, Tonmetta MA, Ames RS, et al. Progressive epitope-blocked panning of a phage library for isolation of human RSA antibodies [J]. Immunol Methods, 2002, 263 (1-2): 123-132.
[61] Rader C, Cheresh DA, Barbas CF. A phage display approach for rapid antibody humanization: designed combinational V gene libraries [J]. Proc. Natl. Acad. Sci. USA, 1998, 95:891-895.
[62] Sblattero D, lou J, Marzari R, et al. In vivo recombination as a tool to generate molecular diversity in phage antibody libraries [J]. Biotechnol, 2001, 74(4):303-315.
[63] Harrd HJ, Neer NV, Reurs A, et al. A large non-immunized human Fab fragment phage library that permit rapid isolation and kinetic analysis of high affinity antibodies [J]. Bio-Chem, 1999, 274(26): 18218-18230.
[64] Gram H, Marconi Let al. In vitro selection and affinity maturation of antibodies from a native combinatorial immunoglobulin library [J]. Proc Natl Acad Sci USA, 1992, 89:3576-3580.
[65] Marks JD. Bypassing immunization:high affinity human antibodies by chain shuffling[J]. Bio/Technology, 1992, 10:779-783.
[66] McCafferty J, Giffith AD et al. Phage antibodies: filamentous phage displaying antibody variable domains [J]. Nature, 1990, 348:552-554.
[67] Wang Gang, Wang Yan, Hua Bing, ea al. Affinity maturation of an anti-keratin human antibodyby chain-shuffling [J]. Chin J Microbiol Immunol, 2001, 21 (2): 160-16.
[68] Yuan Bin, Hou Xiaojun, Wang Hui, et al. Affinity mature of human anti-TNF-α Fab through construction a light chain second library [J]. 2001, 17(2): 179-181.
[69] 王刚,王琰.用硫氰酸盐洗脱法筛选高亲和力噬菌体抗体[J].中国免疫学杂志,2002,18(2):93-95.
[70] Balint RF and Larrick JW. Antibody engineering by parsimonious mutagenesis[J]. Gene, 1993, 137: 109-118.
[71] Deng SJ, Mac Kenzie CR, Narang SA. Simultaneous randomization of antibody CDRs by a synthetic ligase chain reaction strategy[J]. Nucleic Acids Res, 1993, 21:4418.
[72] Deng SJ, Mac Kenzie CR, Hirama T, et al. Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries[J]. Proc Natl Acad Sci USA, 1995, 92:4992.
[73] Chen Y, Wiesmann C, Fuh G, et al. Selection and analysis of optimized anti-VEGF antibody: crystal structure of affinity-matured Fab in complex with antigen[J]. Mol Biol, 1999, 293:865.
[74] 阚劲松.噬菌体抗体库技术研究进展[J].生物技术,2003,13(1):43-45.
[75] Burton DR, Barbas CF Ⅲ, Person MAA, et al. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic individuals Proc Natl Acad Sci USA, 1991; 88:10134.
[76] Barbas CF Ⅲ, Bjorling E, Chiodi F, et al Recombinant Fab fragments neutralize human typel immunodeficiency virus in vitro. Proc Natl Acad Sci USA, 1992; 89:9339.
[77] Barbas CF Ⅲ, Crowe JE, Cababa D, et al. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc Natl Acad Sci USA, 1992; 89:10164.
[78] Barrbas CF. Burbon DR, et al 1993 Cold Spring Harbor Laboratory Course on: Monoclonal antibodies from combinatorial libraries.
[79] 刘晓琳,王刚,化冰.启动子控制严密性对抗体库多样性影响的研究[J].海军总医院学报,2001,14(3):139-143.
[80] Davies E L, Smith J S, Birkett C R, et al. Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes[J]. Immunol Methods,1995, 186(1):125-135.
[81] Andris-Widhopf J, Rader C, Steinberger P, et al. Methods for the generation of chicken monoclonal antibody fragments by phage display [J]. Immunol Methods, 2000, 242(1-2):159-181.
[82] Smith GP, Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985, 228:1315.
[83] 杨安钢,毛积芳,药立波.分子生物学及生物化学技术(M).第1版.北京:高等教育出版社,2001:238—240.
[84] Barbas CF. Lerner RA. et al. Combinatorial immunoglobulin libraries on the surface of phage: rapid selection of antigen-specific Fabs. Methods: A companion to methods in enzymology[J]. 1991, 2(2):119.
[85] J.萨姆布鲁克.E.F.弗里奇等.分子克隆实验指南(第二版).
[2] Kohler G, Mistein. Continuous cultures of fused cells secreting antibody predefined specificity[J]. Nature, 1975, 256:495-513.
[3] Li Haiyan, Xin Xiaoguang, Tian Guobin, et al. Dot-enzyme-linked immunosorbent assay for detection of antibodies to avian influenza virus [J]. Chin J Preventive veterinary medicine. 1999, 21(5):321—324.
[4] 李海燕,于震康,辛晓光,等.禽流感病毒重组核蛋白ELISA诊断技术的研究[J].中国预防兽医学报.2000,22(3):182—185.
[5] Li Haiyan, Yu Kangzhen, Xin Xiaoguang, et al. Development and validation of indirect enzyme-linked immunosorbent assay test kit for detecting anti-influenza virus antibodies [J]. Chin J Preventive Veterinary Medicine. 2001, 23(5):372—376.
[6] 唐秀英,田国斌,于震康,等.禽流感油乳剂灭活疫苗的研究.中国预防兽医学报[J].1999,21(6):401—405.
[7] 罗开健,辛朝安,薛义山,等.禽流感二价抗原油乳剂灭活疫苗研制[J].2000,30(7):3-5.
[8] Suarez DL, Schltz-Cherry S. Immunology of avian influenza virus: a review[J]. Developmental and comparative immunology[J]. 2000, 24:269-283.
[9] Winter G, Giffith AD, Hawkins RE, et al. Making antibodies by phage display technology [J] Annu Rev Immunol, 1994, 12:433-455.
[10] Relph Rapley. The biotechnology and applications of antibody engineering[J]. Molecul Biotechnol, 1995, 3:139-151.
[11] Hoogenboom HR, De Bruine AP, Hufton SE, et al. Antibody phage display technology and its applications [J]. Immunotechnology, 1998, 4:1-20.
[12] Lu J, Sloan SR, An alternating selection strategy for cloning phage display antibodies [J]. Immunol Methods, 1999, 228(1-2): 109-119.
[13] 万英,代佳平,王燕,等.一种稳定、易于操作的噬菌体随机肽库载体的构建[J].第三军医大学学报,1999,21(4):256-258.
[14] Skerra A, Pluckthum A, Assembly of a functional immunoglobulin Fv fragment in Escherichia Coli[J]. Science, 1988, 240:1038.
[15] Better M, Chang CP, Robinson RR, et al. Escherichia Coli secretion of an active chimeric antibody fragment [J]. Science, 1988, 240:1041.
[16] Barbas CF. Kang AS. Et al. Assembly of combinatorial antibody libraries on phage surface: The gene Ⅲ site [J]. Science. USA 1991, 88:7978.
[17] Hoogenboom HK, Griffiths AD. et al. Malti-subunit Proteins on the surface of of filamentous phage methodologies for displaying antibody(Fab) heavy and light chain [J]. Nucleic Acids Res. 1991, 19: 4133.
[18] Jesper LS, Messens JH, Keyser AD, et al. Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene Ⅵ [J]. Bio Technology,1995, 13: 378-382.
[19] Gap C, Map S, Lo CH, et al. Marking artificial antibodies: a format for phage display of combinatorial heterodimeric arrays [J]. Science, 1999, 96(11):6025-6030.
[20] Alting-Mess MA and JM. Polycos vectors:a system for packing filamentous phage and phagemid vectors using lambda phage packing extracts[J]. Gene, 1993, 137:93-100.
[21] Tsurshita N, Fu H and Warren C. Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries[J]. Gene, 1996, 172:59-63.
[22] Den W, Sompuram SR, Sarantopoulos S, et al. A bi-directional phage display vector for the selection and mass transfer of polyclonal antibody libraries[J]. Immunol, Methods 1999, 222:45-57.
[23] Hawkins RE, Russell SJ, Winter G, Selection of phage antibodies by binding affinity mimicking affinity maturation[J]. Mol Biol, 1992,4: 1-20.
[24] Hoogenboom HR, Bruine AP, Hufton SE, et al. Antibody phage display technology and its applications[J]. Immunotechnology, 1998, 4: 1-20.
[25] Tordsson J, Abrahmsen L, Kalland T, et al. Efficient selection of ScFv antibody phage by adsorption in situ expressed antigens in tissue sections[J]. Immunol Methods, 1997, 210: 11-23.
[26] Johns M, George JT, Ritter MA. In vivo selection of ScFv from phage display libraries[J]. Immunol Methods, 2000, 239:137-151.
[27] Osbourn JK, Derbyshire EJ, Vaughan TJ, et al. Pathfinder selection; in situ isolation of novel antibodies, Immunotechnology, 1998, 3: 293-302.
[28] Ward RL, Clark Ma, Lees J, et al. Retrieval of human antibodies from phage-display libraries using enzymatic cleavage[J]. Immunol Methods, 1996, 189: 73-82.
[29] Ridgway JBB, Ng E. Kern JA, et al. Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell line[J]. Cancer Res, 2000, 59:2718-2723.
[30] Ditzel HJ, Binley JM, Moore JP, et al. Neturalizing Recombinant human antibodies to a conformational V2-and CD4 binding site sensitive epitope of HIV-gp120 isolated by using an epitope-masking procedure[J]. Immunol, 1995, 154: 893-906.
[31] 王琰,刘群英,化冰,等.从人源性噬菌体抗体库分离出一株有异常序列的抗HBsAg Fab克隆.中国免疫学杂志[J].1998,14:115-118.
[32] Rojas G, A lmagro JC, Acevedo B, et al. Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable region [J]. J Biotechnol, 2002, 94 (3): 287—298.
[33] 王琰.抗体库技术[J].单克隆抗体通讯,1993,(9):23.
[34] Aujame L, Geoffroy F, Sodoyer. High affinity human antibodies by phage display. Human Antibodies. 1997, 8: 155-168.
[35] 胡建林,郭先健,胡义德,等.肺癌患者噬菌体抗体库的构建及抗肺癌单抗的筛选[J].免疫学杂志,2002,18(4):64-68.
[36] 何进,秦新裕,刘康达,等.胃癌相关的人源单链抗体基因文库的构建[J].实用癌症杂志,2002,17(2):113-115.
[37] Wang Zhiyi, Liu Qi, Wan Zesheng, et al. Construction of human combinatorial immunoglobulinli library containing HbsAb on the surface of phage[J]. Chin J Hepatol, 1999, (7): 159—161.
[38] 余宙耀,粟宽源,任向荣,等.工程化人源性抗HBs Fab抗体的制备、纯化和鉴定[J].上海免疫学杂志.1999,19(2):91—93.
[39] 尤长宣,罗荣城,丁雪梅.基因工程技术制备人源化抗HbsAg抗体Fab片段[J].上海免疫学杂志.2000,20(1):33—37.
[40] 王刚,王琰,化冰.基因工程抗角蛋白抗体的制备与鉴定.海军总医院学报[J].2002,15(1):9—13.
[41] Griffiths A D, Malmqvist M, Marks J D, et al. Human anti-self antibodies with high specificity from phage display libraries. [J]. EMBO J, 1993, 12(2):725-734.
[42] Marks J D, Hoogenboom H R, Bonnert T P, et al. By-passing immunization; human antibodies from V-gene libraries displayed on phage [J]. J Mol Biol, 1991, 222(3):581-597.
[43] de Haard H J, van Neer N, Reurs A, et, al. A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies[J]. J Biol Chem, 1999, 274(26): 18218-18230.
[44] 郭庆东,章翔,费舟,等.大库容量人源性天然单链抗体库的构建[J].第四军医大学学报,2001,22(23):2158-2161.
[45] Wang Gang, Wang Yan, Hua Bing, ea al. Construction of a large naive human antibody librayy [J]. Chin J Cell Mol Immunol, 2002, 18(1): 69-72.
[46] Bender E, Pilkington GR, Burton DR. Human monoclonal Fab Fragments from a combinatorial library prepared from an individual with a low serum titer to a virus [J]. H um A ntbod Hybridanas, 1994;5:2.
[47] Burton DR, Barbas CF Ⅲ, Persson MAA, et al. A large array of human an monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptom atic individual [J]. Proc Natl Acad Sci USA, 1991, 88:10134.
[48] Barbas CF Ⅲ, Crowe JE, Cababa D, et al Human an monoclonal Fab fraqments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein andneutralize infectivity [J]. Proc Natl Acad Sci USA, 1992, 89: 10164.
[49] Aujame L, Geoffroy F, Sodoyer. High affinity human antibodies by phage display[J]. Human Antibodies. 1997, 8: 155-168.
[50] Jespers LS, Roberts S, Mahler SM, et al. Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen[J]. Biotechnology-N-Y, 1994, 12:899.
[51] 刘志刚,俞炜源,林建波,等.人源化鼠抗人纤维蛋白单链抗体的体外成熟[J].细胞与分子免疫学杂志,2001,17(5):462-463.
[52] 程巨龙,王祥斌,张众,等.一种构建改形单域抗体的方法[J].遗传学报,2002,29(3):189-195.
[53] Sheets MD, Amersdorfer P, Finnern R, et al. Efficient construction of a large nonimmune phage antibody library: the production of high affinity human single-chain antibodies to protein antigens[J]. Proc Natl Acad Sci USA, 1998, 95:6157.
[54] Turner DJ, Ritter MA, George AJT, et al. Importance of the linker in expression of single chain Fv antibody fragments: optimization of pepitide sequence using phage display technology[J]. Immunol Methods, 1997,(205):43-54.
[55] Hennecke F, Krebber C, Pluckthum A, et al. Non-repetitive single-chain Fv linkers selectively infective phage(SIP)technology[J]. Protein Engng, 1998,(11):405-410.
[56] 陈晓穗,朱迎春,王欲晓,等.抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达[J].中国免疫学杂志,2000,16(5):248-25.
[57] Coia G, Ayres A, Lilley GG, et al. Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against hepatitis B[J]. Gene, 1997, 201: 203-209.
[58] Saviranta P, Pajunen M, Jauria P, et al. Engineering the steroid-specificity of an anti-17β-estradiol Fab by radom mutagenesis and competitive phage panning[J]. Protein Engng, 1998, 11: 143-152.
[59] Iba Y, Hayashi N, Sawada J, et al. Change in the specificity of antibodies against steroid antigens by introduction of mutation into complementarity-dtermining regions of the V_H domin. [J] Protein Engng, 1998, 11: 361-370.
[60] Tsui P, Tonmetta MA, Ames RS, et al. Progressive epitope-blocked panning of a phage library for isolation of human RSA antibodies [J]. Immunol Methods, 2002, 263 (1-2): 123-132.
[61] Rader C, Cheresh DA, Barbas CF. A phage display approach for rapid antibody humanization: designed combinational V gene libraries [J]. Proc. Natl. Acad. Sci. USA, 1998, 95:891-895.
[62] Sblattero D, lou J, Marzari R, et al. In vivo recombination as a tool to generate molecular diversity in phage antibody libraries [J]. Biotechnol, 2001, 74(4):303-315.
[63] Harrd HJ, Neer NV, Reurs A, et al. A large non-immunized human Fab fragment phage library that permit rapid isolation and kinetic analysis of high affinity antibodies [J]. Bio-Chem, 1999, 274(26): 18218-18230.
[64] Gram H, Marconi Let al. In vitro selection and affinity maturation of antibodies from a native combinatorial immunoglobulin library [J]. Proc Natl Acad Sci USA, 1992, 89:3576-3580.
[65] Marks JD. Bypassing immunization:high affinity human antibodies by chain shuffling[J]. Bio/Technology, 1992, 10:779-783.
[66] McCafferty J, Giffith AD et al. Phage antibodies: filamentous phage displaying antibody variable domains [J]. Nature, 1990, 348:552-554.
[67] Wang Gang, Wang Yan, Hua Bing, ea al. Affinity maturation of an anti-keratin human antibodyby chain-shuffling [J]. Chin J Microbiol Immunol, 2001, 21 (2): 160-16.
[68] Yuan Bin, Hou Xiaojun, Wang Hui, et al. Affinity mature of human anti-TNF-α Fab through construction a light chain second library [J]. 2001, 17(2): 179-181.
[69] 王刚,王琰.用硫氰酸盐洗脱法筛选高亲和力噬菌体抗体[J].中国免疫学杂志,2002,18(2):93-95.
[70] Balint RF and Larrick JW. Antibody engineering by parsimonious mutagenesis[J]. Gene, 1993, 137: 109-118.
[71] Deng SJ, Mac Kenzie CR, Narang SA. Simultaneous randomization of antibody CDRs by a synthetic ligase chain reaction strategy[J]. Nucleic Acids Res, 1993, 21:4418.
[72] Deng SJ, Mac Kenzie CR, Hirama T, et al. Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries[J]. Proc Natl Acad Sci USA, 1995, 92:4992.
[73] Chen Y, Wiesmann C, Fuh G, et al. Selection and analysis of optimized anti-VEGF antibody: crystal structure of affinity-matured Fab in complex with antigen[J]. Mol Biol, 1999, 293:865.
[74] 阚劲松.噬菌体抗体库技术研究进展[J].生物技术,2003,13(1):43-45.
[75] Burton DR, Barbas CF Ⅲ, Person MAA, et al. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic individuals Proc Natl Acad Sci USA, 1991; 88:10134.
[76] Barbas CF Ⅲ, Bjorling E, Chiodi F, et al Recombinant Fab fragments neutralize human typel immunodeficiency virus in vitro. Proc Natl Acad Sci USA, 1992; 89:9339.
[77] Barbas CF Ⅲ, Crowe JE, Cababa D, et al. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc Natl Acad Sci USA, 1992; 89:10164.
[78] Barrbas CF. Burbon DR, et al 1993 Cold Spring Harbor Laboratory Course on: Monoclonal antibodies from combinatorial libraries.
[79] 刘晓琳,王刚,化冰.启动子控制严密性对抗体库多样性影响的研究[J].海军总医院学报,2001,14(3):139-143.
[80] Davies E L, Smith J S, Birkett C R, et al. Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes[J]. Immunol Methods,1995, 186(1):125-135.
[81] Andris-Widhopf J, Rader C, Steinberger P, et al. Methods for the generation of chicken monoclonal antibody fragments by phage display [J]. Immunol Methods, 2000, 242(1-2):159-181.
[82] Smith GP, Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985, 228:1315.
[83] 杨安钢,毛积芳,药立波.分子生物学及生物化学技术(M).第1版.北京:高等教育出版社,2001:238—240.
[84] Barbas CF. Lerner RA. et al. Combinatorial immunoglobulin libraries on the surface of phage: rapid selection of antigen-specific Fabs. Methods: A companion to methods in enzymology[J]. 1991, 2(2):119.
[85] J.萨姆布鲁克.E.F.弗里奇等.分子克隆实验指南(第二版).