Cloning, Expression of the Lectin EGF Domain of P Selectin, and Preparation of Its Monoclonal Antibody

Cloning,
Expression of the Lectin-EGF Domain of P-Selectin,
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 IgG2, IgG1,
and IgG3 respectively, and their light chains were all &kgr;
chain. Immuofluorescence and FACS assays demonstrated that mAbs could
specifically recognize P-selectin expressed on ECV (endothelial cell line)
stimulated by LPS. Meanwhile, the role of mAbs to P-selectin lectin-EGF domain
was studied, and it was proved that the mAbs markedly inhibited adhesion
between platelets and neutrophils in vitro. These monoclonal antibodies
can specifically recognize the natural P-selectin and markedly inhibit adhesion
between platelets and neutrophils in vitro.

Key words P-selectin; domains;
prokaryotic expression; monoclonal antibody; functions

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