Liver Cancer
Copyright ©The Author(s) 2005. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 14, 2005; 11(26): 3985-3989
Published online Jul 14, 2005. doi: 10.3748/wjg.v11.i26.3985
Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library
Bing Yu, Ming Ni, Wen-Han Li, Ping Lei, Wei Xing, Dai-Wen Xiao, Yu Huang, Zhen-Jie Tang, Hui-Fen Zhu, Guan-Xin Shen
Bing Yu, Wen-Han Li, Ping Lei, Wei Xing, Dai-Wen Xiao, Yu Huang, Zhen-Jie Tang, Hui-Fen Zhu, Guan-Xin Shen, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Ming Ni, Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Major State Basic Research Development Program of China, 973 Program, No. 2002CB513100
Correspondence to: Guan-Xin Shen, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. guanxin_shen608@hotmail.com
Telephone: +86-27-83692611 Fax: +86-27-83693500
Received: October 23, 2004
Revised: November 16, 2004
Accepted: November 19, 2004
Published online: July 14, 2005
Abstract

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library.

METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation.

RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted Mr value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells.

CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Keywords: ScFv; Biopanning; HCC; IMAC; Phage display