Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on zymosan A-induced peritonitis in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the inflammation in mice with zymosan A-induced peritonitis.</p><p><b>METHODS</b>In mice with peritonitis induced by intraperitoneal injection of zymosan A, pretreatments with PBS, normal rabbit IgG and TSP-2 antibody at two different doses (2.5 and 5.0 mg/kg) were administered via the tail vein. Six hours after intraperitoneal injection of zymosan A, Evans blue was injected through the tail vein, and the frequency of writhing of the mice within 20 min were recorded. The mice were then sacrificed for peritoneal lavage, and the lavage fluid was collected to assess the exudation of Evans blue in the supernatant. The peritoneal leukocyte count, mast cell degranulation and release of such inflammatory mediators as platelet activating factor (PAF) and tumor necrosis factor-alpha (TNFalpha) in the lavage fluid were observed by cell counting, specific cell staining, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Compared with PBS or rabbit IgG groups, TSP-2 treatment resulted in significantly reduced writhing response of the mice and lowered Evans blue exudation and leukocyte count in the peritoneal lavage, with also decreased degranulation of the mast cells induced by C48/80.</p><p><b>CONCLUSION</b>TSP-2 antibody against a single epitope of mTLR2ECD inhibits the inflammatory response in mice with zymosan A-induced peritonitis.</p>

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

<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>