and Preparation of Its Monoclonal Antibody
ZHOU Tong*, SONG Wei, WANG Feng, NI Pei-Hua, CHEN Nan, ZHANG Dong-Qing1, YU Qi-Wen1
( Department of Nephrology of Ruijin Hospital and
1Shanghai Institute of Immunology, Shanghai Second Medical
University, Shanghai 200025, China )
Abstract To prepare monoclonal antibody specific
to P-selectin lectin-EGF domain, the gene for lectin-EGF domain of P-selectin L-EGF
was amplified from normal human platelets by RT-PCR, then was cloned into
prokaryotic vector pET42b(+). The recombinant plasmid was transformed into E.
coli DH5&agr; strain for further screening and characterization, and
was expressed in E. coli BL21 strain. Expressed protein was purified by
chromatography on a Ni2+-NTA superflow agarose column and eluted
with pH 8.0-4.5 urea gradient. Then the mAb anti-lectin-EGF was prepared with
classical hybridoma technique, and 3 hybridoma cell lines (B10, F3 and H5) were
obtained with Ig subclasses of these mAbs were IgG
Key words P-selectin; domains;
prokaryotic expression; monoclonal antibody; functions
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