title: Generation of AcGFP fusion with single-chain Fv selected from a phage display library constructed from mice hyperimmunized against 5-methyl 2'-deoxycytidine.
abstract: DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.
authors.affiliation: Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Japan.
authors.forename: Motohiro
authors.initials: M
authors.lastname: Ohshima
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Inoue
authors.forename: Hideki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Daiki
authors.initials: D
authors.lastname: Tsuji
authors.forename: Michinao
authors.initials: M
authors.lastname: Mizugaki
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Itoh
mesh_terms: D000818:Animals; D004247:DNA / Q000276:immunology; D019175:DNA Methylation; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000276:immunology; D005260:Female; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D037402:Hydrozoa / Q000235:genetics; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C485192:green fluorescent protein, Aequorea victoria; D003841:Deoxycytidine; D049452:Green Fluorescent Proteins; D004247:DNA; C016569:5-methyldeoxycytidine