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Articles by M Ohshima
Total Records ( 2 ) for M Ohshima
  M Ohshima , K Inoue , H Hayashi , D Tsuji , M Mizugaki and K. Itoh
 

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 (m5dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were contructed 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 light chains. The scFv library was selected against m5Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m5Cyd-BSA. Two scFvs with high reactivity for m5Cyd-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.

  M Ohshima , T Tadakuma , H Hayashi , K Inoue and K. Itoh
 

We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly4-Ser)3. The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m5dCyd and weakly with 5-methylcytidine (m5Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC50 0.054 µg/ml) was ~80 times higher than that of the parent MoAb (IC50 4.27 µg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m5dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained ~1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

 
 
 
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