[Generation and characterization of the monoclonal antibody and scFv against yeast telomeric guanine-quadruplex DNA].
Most eukaryotic telomeres contain many tandem repeats of G-rich sequence. In budding yeast, Sacchromyces cerevisiae, the G-rich sequence can form parallel-stranded quadruplex comformation (G4-DNA) in vitro. Whether this structure exsits in vivo is unknown. To address this question, we generated the antibodies against the G4-DNA of the S. cerevisiae by immunizing the BALB/c mouse with in vitro synthesized G4-DNA oligonucleotides. The antibodies recognize G4-DNA, as well as G-rich DNA sequence in vitro. In order to improve the affinity to G4-DNA substrate, we cloned V(H) and V(L) genes of the antibodies and constructed the single-chain antibody fragment (scFv) with a peptide linker. The recombinated scFv was successfully expressed in E. coli and purified by the affinity chromotography. Based on the sequence of the scFv, we proposed the structure of the antibody by computer-remodeling. The engineered antibodies will be used to detect the existence of the existence of the G4-DNA structure in vivo.
Dong
Qi
Q
Max-Planck Junior Research Group, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D004271:DNA, Fungal / Q000235:genetics / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D054856:G-Quadruplexes; D006147:Guanine / Q000737:chemistry*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016615:Telomere / Q000235:genetics
D000911:Antibodies, Monoclonal; D004271:DNA, Fungal; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D006147:Guanine