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Preparation and identification of the polyclonal antibody against extracellular domain(405T-573Q) of rat Toll-like receptor 2

LIU Li;LAN Xi;LI Jing;YI Jing;WANG Xuan;LI Yue;ZHANG Fujun;L Shemin;LI Dongmin;Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Xi'an Jiaotong University;  
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 p ET28a-TLR2 ex 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 p ET28a-TLR2 ex 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.
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