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Preparation and Specificity Analysis of scFv Aganinst Salmonella enteritidis |
WANG Bao-gui1, WU Xiao-li2, DONG Su-qin1, GAN Min1, CHEN Xing-xing1, CHEN Fei1, MING Xing1, XU Feng1 |
1. Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China; 2. Jiangxi University of TCM International Education College, Nanchang 330004, China |
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Abstract Objective: To obtain the specific scFv against Salmonella enteritidis using genetic engineering technology. Methods: VH and VL genes were amplified by Reverse Transcription (RT) PCR from hybridoma cells secreting anti-Salmonella enteritidis monoclonal antibody, scFv gene (VL-Linker-VH) was obtained by Splice Overlap Extension (SOE) PCR with flexible polypeptide Linker connector (Gly4Ser)3. Subsequently the scFv-pGEX-4T-1 recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was purified by GST chromatography and identified by SDS-PAGE and ELISA. Results: DNA sequencing demonstrated that scFv was successfully inserted into pGEX-4T-1. SDS-PAGE demonstrated that the molecular weight of the expressed scFv was about 60 kDa, and ELISA results confirmed that the obtained scFv can be recognized by not only Salmonella enteritidis but also S. enterica subsp enterzca, S. anatum and S. typhimurium. Conclusion: Specific scFv against S. enteritidis was obtained and can be used as candidate antibody molecules in detection of Salmonella.
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Received: 12 November 2012
Published: 25 May 2013
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[1] 杨保伟, 张秀丽, 曲东, 等. 2007-2008 陕西部分零售畜禽肉沙门氏菌血清型和基因型. 微生物学报,2010,(5):654-660. Yang B W, Zhang X L, Qu D, et al.Salmonella serotypes and genotypes of retail poultry meat from shanxi Province in 2007-2008.Journal of Microbiology,2010,(5):654-660. [2] 朱超, 许学斌. 沙门菌属血清型诊断. 上海: 同济大学出版社, 2009,12-140. Zhu Ch, Xu X W. Salmonella Serotypes Diagnosis. Shanghai:Tongji University Press, 2009,12-140. [3] 赵贵, 张华. 畜产品中沙门氏菌的危害及检测方法概述. 贵州畜牧兽医,2004,28(3):21-22. Zhao G, Zhang H. Overview of hazards and detection of Salmonella in animal products. Animal Husbandry and Veterinary, 2004,28(3):21-22. [4] 何晓青. 沙门氏菌检验技术进展. 中国公共卫生, 2003,19(10):1260-1262. He X Q.Assay Technical Progress of Salmonella. Chinese Journal of Public Health, 2003,19(10):1260-1262. [5] 黄金海, 孙跃辉, 陈瑞, 等. 食品中沙门氏菌 LAMP 快速检测方法的建立. 天津大学学报. 2012,45(5):468-472. Huang J H, Sun Y H, Chen R, et al. LAMP method for rapid detection Salmonella in food.Learned Journal of Tianjin University. 2012,45(5):468-472. [6] Huston J S, Levinson D, Mudgett-Hunter M, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences, 1988,85(16):5879-5883. [7] Bird R E, Hardman K D, Jacobson J W, et al. Single-chain antigen-binding proteins. Science (New York, NY), 1988,242(4877):423. [8] Joosten V, Lokman C, Van Den Hondel C A, et al. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. Microbial Cell Factories, 2003,2(1):1. [9] 沈倍奋, 陈志南, 刘民培. 重组抗体.北京:科学出版社,2005.18-117,217-245. Chen B F, Chen ZH N, Liu M P. Recombinant Antibody. Beijing: Science Press, 2005.18-117,217-245. [10] 刘怀田, 黄策. 重组人宋内痢疾杆菌脂多糖抗独特型抗体重链可变区基因的高效表达及纯化.中华微生物学和免疫学杂志,1997,17(6):453-456. Liu H T, Huang C. Expression and purification of anti-idiotype antibody heavy chain variable region gene from recombinant human Shigella sonnei lipopolysaccharide. Chinese Journal of Microbiology and Immunology, 1997, 17(6):453-456. [11] Ward E S, Ggussow D, Ggriffths A D,et al. Binding of arepertoire of single immunoglbuLin Variable domains secretedform schcerzchza colz. Natmre, 1989, 34(1): 544-546. [12] 秦丽莉, 张春明. 单链抗体研究进展及其在医学中的应用. 国外医学: 放射医学核医学分册,2006,29(6):255-257. Qin L L, Zhang CH M, Progress in Research on Single-chain Antibody and Its Medical Applications. Foreign Medical: Radiation Medicine and Nuclear Medicine, 2006, 29(6):255-257. [13] 石凌超, 韩红辉, 唐喆伟, 等. 抗苏丹红单链 Fab 抗体的构建, 表达及功能鉴定. 现代免疫学, 2009,(004):274-278. Shi L Ch, Han H H, Tang J W, et al. Anti-Sudan single chain antibody Fab Construction, expression and identification. Modern Immunology, 2009,(004):274-278. |
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