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ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(10): 707–712
CN 31-1940/Q
Rapid Purification of a New Humanized Single-chain Fv Antibody/Human
Interleukin-2 Fusion Protein Reactive against HER2 Receptor
Wei-Yun ZHANG*, Tak-Chun YIP1,
and Cheuk-Sang KWOK2
Medical School,
Nanjing University, Nanjing 210093, China;
1Department of
Clinical Oncology, Queen Elizabeth Hospital, Hong Kong, China;
2Division of
Radiation Oncology, City of Hope National Medical Center, Duarte, CA91010, USA
Abstract Human embryonic kidney
293 cells were transfected with plasmid pcDNA-H520C9scFv-rhIL-2 containing a
chimeric cDNA encoding the humanized 520C9 scFv/recombinant human IL-2 fusion
protein (H520C9scFv-rhIL-2). The transfected cells in plateau growing phase
were cultured in serum-free medium for three days. The supernatant was
collected, concentrated and purified using an affinity column packed with
CNBr-activated Sepharose 4B coupled with anti-rhIL-2 mouse monoclonal antibody.
The purified fusion protein was analyzed by ELISA, SDS-PAGE and Western blot.
The fusion protein showed only one band in both silver stained electrophoresis
gel and Western blot developed by ECL chemiluminescence system. Its molecular
weight was confirmed to be about 45 kD. This fusion protein possessed binding
specificity against p185 positive SKOV3 and B16/neu cells, and it might
stimulate IL-2-dependent CTLL-2 cell proliferation as well.
Key words single-chain Fv antibody; IL-2; fusion protein; human embryonic
kidney 293 cells; affinity chromatography
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Received: June 1, 2004 Accepted: August 16,
2004
This study was supported by a grant from the Hong Kong Universities
Research Council, and a Pilot Study grant from the Hong Kong Polytechnic
University
*Corresponding
author: Tel, 86-25-83686149; E-mail, [email protected]