Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To prepare the recombinant murine Toll-like receptor-2 N-terminal (mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.</p>