Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody.
- Author:
Cui-lan YANG
1
;
Wen-zhong ZHAO
;
Yan-jun LIU
;
Ping ZHU
;
Ning FU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies, Monoclonal; biosynthesis; CHO Cells; Cell Line; Cloning, Molecular; Cricetinae; Cricetulus; Enzyme-Linked Immunosorbent Assay; Escherichia coli; genetics; Genetic Vectors; Immune Sera; immunology; Immunohistochemistry; Mice; Rabbits; Recombinant Fusion Proteins; biosynthesis; immunology; isolation & purification; Toll-Like Receptor 2; biosynthesis; genetics; immunology; Transfection
- From: Journal of Southern Medical University 2006;26(11):1609-1615
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo prepare the recombinant murine Toll-like receptor-2 N-terminal (mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody.
METHODSThe gene encoding 153 amino acids of mTLR-2N was amplified by PCR and cloned into pET32A vector with sequence verification. The recombinant fusion protein was expressed in E. coli and purified by Probond resin column. Rabbits were immunized with fusion protein to obtain the polyclonal anti-sera, and the antibodies were identified by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.
RESULTSThe recombinant fusion protein was efficiently expressed and purified. The polyclonal antibodies could bind to the fusion protein expressed in different vectors as the antigens in ELISA, and also bind with RAW264.7 cells expressing mTLR-2 and CHO cells transfected with full-length mTLR-2 gene.
CONCLUSIONThe recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.
