[Preparation and Identification of the Polyclonal Antibody Against Extracellular Domain (405T-573Q) of Rat Toll-like Receptor 2].

OBJECTIVE:To prepare the polyclonal antibody of the extracellular domain (405T-573Q) of rat Toll-like receptor 2 (TLR2) and identify its activity.METHODS:Firstly, the extracellular domain (507 bp) of rat TLR2 was amplified from rat hepatocytes by reverse transcription PCR, and the recombinant plasmid pET28a-TLR2ex was constructed. Secondly, the fusion protein TLR2ex-6×His was expressed in E.coli BL21(DE3) and purified by affinity chromatography. And then, the polyclonal antibody was prepared by immunizing the BALB/c mice with the purified fusion protein. Finally, the titer and specificity of the polyclonal antibody were analyzed by ELISA and Western blotting, respectively.RESULTS:The recombinant plasmid pET28a-TLR2ex was successfully constructed, and the TLR2ex-6×His fusion protein was successfully expressed and purified. Western blotting demonstrated that the antibody from the TLR2ex-6×His-immunized BALB/c mice specifically and selectively bound with rat TLR2. ELISA also indicated that the titer of polyclonal antibody reached 1:76 800.CONCLUSION:The polyclonal antibody against the extracellular domain (405T-573Q) of rat TLR2 has been successfully prepared.