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KMID : 1161520140180040259
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Animal Cells and Systems
2014 Volume.18 No. 4 p.259 ~ p.266
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Production of an epitope-specific antibody using recombinant repetitive oligonucleotides
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Park Sang-Yul
Kim Hyun-Jeong
Seo Ja-Young
Choi Eui-Yul
Oh Sang-Wook
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Abstract
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This study was performed to produce a strong antibody against repeated peptides for a specific region of an antigen. Nine amino acids at C-terminal from human brain β-tubulin III were selected as a target sequence since it is well known for a high conservancy of sequence and resistance to antibody production. The synthetic oligonucleotides had six bases (5′-CGAC?GT-3′, 5′-TC?CAGC-3′) appended at each end and were repeatedly linked and directly joined by ligase in the presence of restriction enzymes (AccI, AvaI) to allow for the construction of head-to-tail repeats. Ligated oligonucleotides containing more than 10-unit repeats were eluted from agarose gels and cloned into the AccI site of a modified pGEX expression vector. The cloned repeated target sequences were subsequently expressed in a bacterial system. A large amount of glutathione-S-transferase-fusion protein containing the unit repeats was purified by affinity chromatography on glutathione-agarose beads and injected into Balb/c mice to produce polyclonal and monoclonal antibodies (Abs). Total protein from several mammalian brains was extracted and utilised to test the quality of the generated Abs in immunoblot assays. The generated Abs specifically recognised the 55-kDa human brain β-tubulin protein but not any other mammalian brain β-tubulins. The potential uses of this system could be applied to an effective epitope generation for an antibody and to the development of a new type of subunit vaccine.
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KEYWORD
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head-to-tail repeat, human brain β-tubulin III, immunogen, monoclonal antibody, repetitive oligonucleotide
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