[Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody].
OBJECTIVE
To prepare the recombinant murine Toll-like receptor-2 N-terminal (mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody.


METHODS
The 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.


RESULTS
The 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.


CONCLUSIONS
The recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.
Yang
Cui-lan
CL
Department of Immunology, Southern Medical University, Guangzhou 510515, China. ycllb@fimmu.com
Zhao
Wen-zhong
WZ
Liu
Yan-jun
YJ
Zhu
Ping
P
Fu
Ning
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D051195:Toll-Like Receptor 2 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D014162:Transfection
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2