Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma.
BACKGROUND
Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques.


RESULTS
The sequences encoding the variable regions of heavy (VH) and light (VL) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab).


CONCLUSIONS
This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.
Sandee
Duanpen
D
Department of Biotechnology, Graduate school of Engineering, Osaka University, Suita, Osaka 565-0871, Japan. duanpensandee@hotmail.com
Tungpradabkul
Sumalee
S
Tsukio
Manae
M
Imanaka
Tadayuki
T
Takagi
Masahiro
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000627:therapeutic use; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017510:Protein Folding; D057127:Single-Chain Antibodies; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; C478634:Hep27scFv; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies