[Cloning, expression of the lectin-EGF domain of P-selectin, and preparation of its monoclonal antibody].
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 alpha strain for further screening and characterization, and was expressed in E. coli BL21 strain. Expressed protein was purified by chromatography on a Ni(2+)-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(2), IgG(1), and IgG(3) respectively, and their light chains were all kappa 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.
Zhou
Tong
T
Department of Nephrology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China. zhoutong_cn@hotmail.com
Song
Wei
W
Wang
Feng
F
Ni
Pei-Hua
PH
Chen
Nan
N
Zhang
Dong-Qing
DQ
Yu
Qi-Wen
QW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D000918:Antibody Specificity; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D037102:Lectins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D019007:P-Selectin / Q000235:genetics* / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D037102:Lectins; D019007:P-Selectin; D004815:Epidermal Growth Factor