噬菌体展示抗克伦特罗抗体文库的构建及单克隆抗体的筛选

生物技术通报 ›› 2014, Vol. 0 ›› Issue (2): 136-142.

噬菌体展示抗克伦特罗抗体文库的构建及单克隆抗体的筛选

董金华¹, 唐玉海¹, 乔宁¹, 韩金宏¹, 董美华¹, 董益阳²

1. 潍坊科技学院生物工程研发中心，山东 262700；2.北京化工大学生命科学与技术学院，北京 100029

收稿日期:2013-08-12 出版日期:2014-02-27 发布日期:2014-02-27
作者简介:董金华，男，副教授，研究方向：生物学；E-mail：newbio2010@gmail.com
基金资助:
潍坊科技学院校级课题（博士基金）（W13K007）

Construction of a Phage-displayed Anti-clenbuterol Antibody Library and Selection of Monoclonal Antibody

Dong Jinhua¹, Tang Yuhai¹, Qiao Ning¹, Han Jinhong¹, Dong Meihua¹, Dong Yiyang²,

（1. Department of Research and Development，Weifang University of Science and Technology，Shandong 262700；2. College of Life Science and Technology，Beijing University of Chemical Technology，Beijing 100029）

Received:2013-08-12 Published:2014-02-27 Online:2014-02-27

摘要/Abstract

摘要： 利用小白鼠免疫和噬菌体展示技术构建了噬菌体展示抗体文库，从文库中筛选获得了一株抗克伦特罗单克隆抗体。该抗体的氨基酸序列尚未在世界基因数据库中登录，是一个新的抗体。该抗体对于游离克伦特罗的半抑制率IC₅₀为0.602 μg/mL。该噬菌体展示的抗体片段可以用于竞争法检测克伦特罗的含量，其能够检测出的最低浓度为15.6 ng/mL，具有较大的实用价值。

关键词: 克伦特罗, 抗体, 噬菌体展示, 免疫检测

Abstract: An anti-clenbuterol antibody was developed by immunization of mouse and construction of a phage displaying antibody library. The antibody has a novel sequence and the utilization of gene segments in germline was also identified. With this antibody, a competitive assay was performed and gave a limit of detection of 15.6 ng/mL. The IC₅₀ of phage-antibody with free clenbuterol against immobilized bovine serum albumin conjugated clenbuterol was 0.602 μg/mL. All the results suggested that the antibody is useful in practical applications.

董金华, 唐玉海, 乔宁, 韩金宏, 董美华, 董益阳. 噬菌体展示抗克伦特罗抗体文库的构建及单克隆抗体的筛选[J]. 生物技术通报, 2014, 0(2): 136-142.

Dong Jinhua, Tang Yuhai, Qiao Ning, Han Jinhong, Dong Meihua, Dong Yiyang,. Construction of a Phage-displayed Anti-clenbuterol Antibody Library and Selection of Monoclonal Antibody[J]. Biotechnology Bulletin, 2014, 0(2): 136-142.

参考文献

[1] Anderson GE, Wilkins A. trial of clenbuterol in bronchial asthma
[J]. Thorax, 1977. 32（6）: 717-719.
[2] Maltin CA, Delday MI, Reeds PJ. The effect of a growth promoting drug, clenbuterol, on fibre frequency and area in hind limb muscles from young male rats[J]. Biosci Rep, 1986. 6（3）: 293-299.
[3] Brockway JM, MacRae JC, Williams PE. Side effects of clenbuterol as
a repartitioning agent[J]. Vet Rec, 1987, 120（16）: 381-383.
[4] Sblattero D, et al. One-step cloning of anti tissue transglutaminase scFv from subjects with celiac disease[J]. J Autoimmun, 2004, 22（1）: 65-72.
[5] Weisser NE, Hall JC. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics[J]. Biotechnol Adv, 2009, 27（4）: 502-520.
[6] Winter G, Griffiths AD, Hawkins RE, et al. Making antibodies by phage display technology[J]. Annual Review of Immunology, 1994. 12: 433-455.
[7] Holt LJ, Büssow K, Walter G, Tomlinson IM. By-passing selection: direct screening for antibody-antigen interactions using protein arrays[J]. Nucleic Acids Res, 2000, 28（15）:E72.
[8] Abbas AK, Sen R. The activation of lymphocytes is in their CARMA
[J]. Immunity, 2003. 18（6）: 721-722.
[9] Engvall E, Jonsson K, Perlmann P. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes[J]. Biochim Biophys Acta, 1971, 251（3）: 427-434.
[10] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975, 256（5517）: 495-497.
[11] Lamminmaki U, Paupério S, Westerlund-Karlsson A, et al. Expanding the conformational diversity by random insertions to CDRH2 results in improved anti-estradiol antibodies[J]. J Mol Biol, 1999, 291（3）: 589-602.
[12] Parhami-Seren B, Viswanathan M, Margolies MN. Selection of high affinity p-azophenyarsonate Fabs from heavy-chain CDR2 insertion libraries[J]. J Immunol Methods, 2002, 259（1-2）: 43-53.
[13] Little M, Welschof M, Braunagel M, et al. Generation of a large complex antibody library from multiple donors[J]. J Immunol Methods, 1999, 231（1-2）: 3-9.
[14] Abe R, Ohashi H, Lijima I, et al. “Quenchbodies”：quench-based antibody probes that show antigen-dependent fluorescence[J]. J Am Chem Soc, 2011, 133（43）: 17386-17394.
[15] Morizane SFY, Makisaka N. Protein biomarker measurement and simple/rapid diagnostics with supersensitive and multiplex assay, MUSTag technology[J]. Rinsho Byori, 2009, 57（11）: 1104-12.
[16] Dong J, Hasan S, Fujioka Y, et al. Detection of small molecule diagnostic markers with phage-based open-sandwich immuno-PCR[J]. J Immunol Methods, 2012, 377（1-2）: 1-7.