Abstract:Humanized monoclonal antibodies against enterovirus type 71 (EV71) are ideal options in clinical treatment of EV71 infections. In this study, variable region genes of human-origin immunoglobulin were amplified to construct recombinant Fab fragments against EV71. Total RNA was extracted from peripheral blood lymphocytes of a normal adult that was positive with EV71 neutralizing antibody. With the reverse-transcribed cDNAs as templates, variable region genes of light and heavy chains were amplified by human IgG antibody primer sets, and the expression cassettes for Fab fragments were subsequently constructed by splicing by overlap extension PCR (SOE-PCR). It was indicated by sequence analysis of obtained 1 500 bp amplicons that diversity in Fab fragments was 94.12%, suggesting successful construction of a Fab fragment expression library, which facilitated preparation of human-origin monoclonal antibody library against EV71.
余艳琼,翁育伟,严延生. 抗EV71病毒人源抗体Fab段表达基因的构建及序列分析[J]. 中国人兽共患病学报, 2014, 30(3): 234-238.
YU Yan-qiong,WENG Yu-wei,YAN Yan-sheng. Construction and sequence analysis of expression cassette for Fab fragments of humanorigin immunoglobulin against EV71. Chinese Journal of Zoonoses, 2014, 30(3): 234-238.
[1] Chatproedprai S, Theanboonlers A, Korkong S, et al. Clincal and molecular characterization of hand-foot-and-mouth disease in Thailand, 2008-2009[J]. Jpn J Infect Dis, 2010, 63(4): 229-233. [2]Li H, Li W. Research progress of enterovirus 71 vaccine[J]. Progt Microbiol Immunol, 2012, 40(2): 89-94. (in Chinese) 李欢, 李薇.肠道病毒71型的研究进展[J].微生物学免疫学进展, 2012 , 40(2): 89-94. [3]Song HJ. Construction and identification on the therapeutic chimeric antibodies with broad-spectrum neutralization to highly pathogenic avian influenza H5N1 virus[D]. Xiamen: Xiamen University, 2009. (in Chinese) 宋慧娟. 高致病性禽流感病毒H5N1治疗性嵌合抗体研究[D].厦门:厦门大学, 2009. [4] Robert R, Lefranc MP, Ghochikyan A, et al. Restricted V gene usage and VH/VL pairing of mouse humoral response against the N-tenninal immunodominant epitope of the amyloidβpeptide[J]. Mol Immunol, 2010, 48(1/3): 59-72. DOI: 10.1016/j.molimm.2010.09.012 [5]Andris-Widhopf J, Steinberger P, Fuller R, et al. Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences[J]. Cold Spring Harbor Protocols, 2011, 2011(9): 1151-1165. DOI: 10. 1101/pdb. prot065565 [6]National Polio and Measles Laboratory. Hand-foot-and-mouth Disease Laboratory Manual[R]. Beijing: Chinese Center for Disease Control and Prevention, 2010: 20-26. (in Chinese) 国家脊髓灰质炎和国家麻疹实验室.手足口病实验室手册[R].北京: 中国疾病预防控制中心,2010: 20-26. [7]Kohler G, Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity[J]. Nature, 1975, 256(5517): 495-497. [8]Chen LH, Song HF, Liu XW, et al. Advances in humanized monoclonal antibody for therapeutic uses[J]. Chin J New Drugs, 2004, 13(7): 590-594. (in Chinese) 陈立慧, 宋海峰, 刘秀文, 等. 治疗性人源化单克隆抗体研究进展[J]. 中国新药杂志, 2004, 13(7): 590-594. [9]Fox KM, Dias JA, Van Roey P. Three-dimensional structure of human follicle-stimulating hormone[J]. Mol Endocriol, 2001, 15(3): 378-389. DOI: 10.1210/me.15.3.378 [10]Furuse M, Hata M, Furuse K, et al. Claudin-based tightjunctions are crucial for the mammalian epidermal barrier: alesson from claudin-deficient mice[J]. J Cell Biol, 2002, 156(6): 1099-1111. DOI: 10.1083/jcb.200110122 [11]Liu XY, Zhao ZF, Xu J, et al. The construction of human phage display library of Alzheimer’s disease[J]. Chin J Gerontol, 2011, 17(31): 3294-3297. (in Chinese) 苏雪莹, 赵振富, 徐杰, 等. 人源性老年痴呆单链噬菌体抗体库的构建[J]. 中国老年学杂志, 2011, 17(31): 3294-3297. [12]Sun LN, Zhang L, Zhang FS, et al. Generation of recombinant human antibodies for EV71 virus[J]. Chin J Exp Clin Virol, 2011, 25(3): 161-163. (in Chinese) 孙丽娜, 张黎, 张福顺, 等. 人源抗EV71病毒基因工程抗体的研究[J]. 中华实验和临床病毒学杂志, 2011, 25(3): 161-163. [3]Flanagan KL, Burl S, Lohman-Payne BL, et al. The challenge of assessing infant vaccine responses in resource-poor settings[J]. Expert Rev Vaccines, 2010, 9(6): 665-674. DOI: 10.1586/erv.10.41 [14]Pallars N, Lefebvre S, Contet V, et al. The human immunoglobulin heavy variable genes[J]. Exp Clin Immunogenet, 1999, 16: 36-60. [15]Gorny MK, Wang XH, Williams C, et al. Preferential use of the VH5-51gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1[J]. Mol Immunol, 2009, 46: 917-926. DOI: 10.1016/j.molimm.2008.09.005 [16]Shao HX, Zhang L, Yang B, et al. Construction and diversity analysis of a naive human phage-displayed Fab library[J]. Chin J Immunol, 2010, 11(10): 1007-1015. (in Chinese) 邵红霞, 章黎, 杨彬, 等. 天然人源IgG Fab噬菌体抗体库的构建及多样性分析[J]. 中国免疫学杂志, 2010, 11(10): 1007-1015. [17]Davidkova G, Pettersson S, Holmberg D, et al. Selective usage of VH genes in adult human B lymphocyte repertoires[J]. Scand J Immunol, 1997, 45: 62-73. DOI: 10.1046/J.1365-3083.1997.d01-376.x