Prokaryotic expression of poTLR3extracellular domain and preparation of polyclonal antibody

In order to preparation polyclonal antibody of porcine toll-like receptor 3(poTLR3) extracellular domain proteins,the extracellular domain of poTLR3 was amplified from the constructed plasmid pGEM-Teasy-poTLR3 and cloned into the prokaryotic expression vector pGEX-4T-1,the recombinant plasmid pGEX-4T-poTLR3 was transformed into BL21 to express fusion protein GST-poTLR3 under the induction of IPTG.Moreover,the GST-poTLR3 fusion proteins were purified,and the antiserum was made by the immunized rabbits.Furthermore,the titer of the polyclonal antibody was tested by the indirect ELISA,and specificity of the antibody was identified by western blot and immunofluorescence.The results indicated that the titer of the polyclonal antibody was up to 1∶128 000,and the antibody combined specifically with the expressed protein of poTLR3 in eukaryotic cell.Therefore,it was showed that the poTLR3 extracellular domain protein was highly expressed in BL21,and the antiserum with high titers and specificities was prepared,and which provides foundation for further investigation in poTLR3 function.