Please use this identifier to cite or link to this item:
標題: 重組抗-氯黴素單鏈抗體之製備與分析
Preparation and characterization of recombinant single-chain variable fragment (scFv) against chloramphenicol
作者: 黃郁涵
Yu-Han Huang
關鍵字: 氯黴素;單鏈抗體;chloramphenicol;single-chain variable fragment;scFv
引用: [1]中華藥典第六版 p340-341 [2]「動物用藥殘留標準」 [3]行政院公報 第019 卷 第149 期 20130809農防字第1021476331A 號 [4] Ehrlich J, Bartz QR, Smith RM, Joslyn DA, Burkholder PR. Chloromycetin, a New Antibiotic From a Soil Actinomycete. Science. 1947; 106(2757):417. [5] Gottlieb D, Carter He, Legator M, Gallicchio V. The biosynthesis of chloramphenicol. I. Precursors stimulating the synthesis. J Bacteriol. 1954; 68(2):243-251. [6] Loren M. Long , H. D. Troutman. Chloramphenicol1 (Chloromycetin). VII. Synthesis through p-Nitroacetophenone. J. Am. Chem. Soc. 1949; 71(7):2473–2475. [7] Chloramphenicol'. The Merck Manual. Merck. [8] Kuni CM, Glazko AJ, Finland M. Persistence of antibiotics in blood of patients with acute renal failure. II. Chloramphenicol and its metabolic products in the blood of patients with severe renal disease or hepatic cirrhosis. J Clin Invest. 1959; 38:1498 – 1509 [9] Friedman CA, Lovejoy FC, Smith AL. Chloramphenicol disposition in infants and children. J. Pediatr. 1979; 95:1071-1077 [10] Girardi C, Odore R. Pharmacological treatments and risks for the food chain. Vet Res Commun. 2008; 32 Suppl 1:S11-8. [11] Turton JA, Fagg R, Sones WR, Williams TC, Andrews CM. Characterization of the myelotoxicity of chloramphenicol succinate in the B6C3F1 mouse. Int J Exp Pathol. 2006; 87(2):101-112. [12] Jimenez JJ, Arimura GK, Abou-Khalil WH, Isildar M, Yunis AA. Chloramphenicol-induced bone marrow injury: possible role of bacterial metabolites of chloramphenicol. Blood. 1987; 70(4):1180-1185. [13] Milhaud, G., Metabolic study discussion on chloramphenicol WHO report, 1983. [14] Pooja Shukla, F. W. Bansode and R. K. Singh. Chloramphenicol Toxicity: A Review. Journal of Medicine and Medical Sciences. 2011; 2(13):1313-1316 [15] Girardi C1, Odore R. Pharmacological treatments and risks for the food chain. Vet Res Commun. 2008; 32 Suppl 1:S11-8. [16] Nemazee D. Receptor editing in lymphocyte development and central tolerance. Nat Rev Immunol. 2006; 6(10):728-740. [17] Woof JM, Burton DR. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol. 2004; 4(2):89-99. [18] Natsume A1, Niwa R, Satoh M. Improving effector functions of antibodies for cancer treatment: Enhancing ADCC and CDC. Drug Des Devel Ther. 2009; 21;(3)7-16. [19] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975; 256(5517):495-497. [20] Steinitz M. Three decades of human monoclonal antibodies: past, present and future developments. Hum Antibodies. 2009; 18(1-2):1-10. [21] Chu-Chen Cheng, Kuan-Huei Hsieh, Yi-Chih Lei, Yung-Te Tai, Tong-Hsuan Chang, Shi-Yuang Sheu, Wen-Ren Li, And Tzong-Fu Kuo. Development and Residue Screening of the 2 Furazolidone Metabolite, 3-Amino-2-oxazolidinone (AOZ), in Cultured Fish by an Enzyme-Linked Immunosorbent Assay. J Agric Food Chem. 2009; 57(13):5687-5692 [22] Yu FY1, Vdovenko MM, Wang JJ, Sakharov IY. Comparison of Enzyme-Linked Immunosorbent Assays with Chemiluminescent and Colorimetric Detection for the Determination of Ochratoxin A in Food. J Agric Food Chem. 2011; 59(3):809–813 [23] Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988; 85(16):5879-5883. [24] Coloma MJ, Hastings A, Wims LA, Morrison SL. Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. Immunol Methods. 1992; 152(1):89-104. [25] Li J, Wang Y, Li QX, Wang YM, Xu JJ, Dong ZW. Cloning of 3H11 mAb variable region gene and expression of 3H11 human-mouse chimeric light Chain. World J Gastroenterol. 1998; 4(1):41-44.. [26] Miller BR Demarest SJ, Lugovskoy A, Huang F, Wu X, Snyder WB, Croner LJ, Wang N, Amatucci A, Michaelson JS, Glaser SM. tability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel. 2010; 23(7):549-557. S [27] Peipp M, Saul D, Barbin K, Bruenke J, Zunino SJ, Niederweis M, Fey GH. Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications. J Immunol Methods. 2004; 285(2):265-280. [28] Serebrovskaya EO, Edelweiss EF, Stremovskiy OA, Lukyanov KA, Chudakov DM, Deyev SM. Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein. Proc Natl Acad Sci U S A. 2009; 106(23):9221-9225. [29] Yuan Y. A survey and evaluation of population-based screening for gastric cancer. Cancer. Biol Med. 2013; 10(2): 72–80. [30] Yan J, Gu GJ, Jost C, Hammond M, Pluckthun A, Landegren U, Kamali-Moghaddam M. A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis. PLoS One. 2014; 9(9): e108061 [31] Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z, Kingsmore SF, Lizardi PM, Ward DC. Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection. Proc Natl Acad Sci U S A. 2000; 97(18): 10113–10119 [32] Pourhassan-Moghaddam M, Rahmati-Yamchi M, Akbarzadeh A, Daraee H, Nejati-Koshki K, Hanifehpour Y, Joo SW. Protein detection through different platforms of immuno-loop-mediated isothermal amplification. Nanoscale Res Lett. 2013; 8(1): 485 [33] Ambekar CS, Cheung B, Lee J, Chan LC, Liang R, Kumana CR. Metabolism of chloramphenicol succinate in human bone marrow. Eur J Clin Pharmacol. 2000; 56(5):405-409. [34] Li J, Wang Y, Li QX, Wang YM, Xu JJ, Dong ZW. Cloning of 3H11 mAb variable region gene and expression of 3H11 human-mouse chimeric light Chain. World J Gastroenterol. 1998; 4(1):41-44.
在食品安全議題受到重視的現今,利用有效且不昂貴的免疫方法快速檢測抗生素殘留以取代昂貴費時的LC-MS分析應更具有發展價值。 因此開發一個高感度的抗體並研製成檢測套組成為確保食品安全的重要方針。
本研究自表現CAP抗體之融合瘤細胞中成功地取得其可變區基因並進一步建構成融合His-tag之單鏈抗體之表現質體,在最適化之大腸桿菌表現宿主BL21(DE3)pLysS表現後可利用Ni-resin親和性管柱純化並獲得CAP之單鏈抗體。利用ELISA方法分析可證實CAP單鏈抗體可以專一識別CAP,在抗體競爭性試驗同時也證實了CAP單鏈抗體與其單株抗體均會競爭相同結構。在未來更可利用易錯聚合酶鏈反應(error prone PCR)來增加靈敏度並輔以融合不同螢光蛋白或結合其他偵測技術以增加免疫檢測的偵測極限與多元應用性。

Chloramphenicol (CAP) is a potent and efficient antibiotic widely used in pharmacological treatments in veterinary and human. Despite being highly effective, it shows severe toxicity as the residual CAP results in Aplastic anemia (AA) and bone marrow suppression. Due to the potential risk in public health of CAP utilization, the use in food-producing animal's therapy has been banned. Taiwan government has established a zero tolerance policy to CAP in livestock products.
Recently, the food crises have grabbed huge public attention in Taiwan. Replacing the expensive LC-MS analysis by cheap and efficient rapid immunoassay to detect the residual illegal compound can better fit to public interest. Therefore, to generate high sensitivity antibodies for immunoassay kits play a key role in improving the food safety.
This study has successfully cloned CAP-scFv gene from hybridoma and constructed CAP-scFv gene onto an expression vector fused with a His-tag gene. The expression of this protein in E. coli BL21(DE3) pLysS was optimized and the expressed recombinant CAP-scFv protein was purified with Ni-IMAC. We confirmed the specificity between CAP-scFv and chloramphenicol via ELISA assay. Furthermore we also demonstrated that CAP-scFv recognizes the same molecular structure as CAP monoclonal antibody does by performing the competition assay with CAP monoclonal antibody. In the future, using the error-prone PCR approach further improve the CAP-scFv sensitivity and fusion of fluorescent to the protein flank on scFv or combination with various detection methods will be the next target to enhance the detection limit of immunoassay and create novel applications to numerous aspects.
Rights: 同意授權瀏覽/列印電子全文服務,2018-02-02起公開。
Appears in Collections:生物化學研究所

Files in This Item:
File Description SizeFormat Existing users please Login
nchu-104-7101058021-1.pdf5.53 MBAdobe PDFThis file is only available in the university internal network    Request a copy
Show full item record
TAIR Related Article

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.