Preparation and application of polyclonal antibody against mouse IL-1α

AIM: To construct a recombinant plasmid encoding mouse IL-1α(mIL-1α),express and purify mIL-1α protein,and prepare its polyclonal antibody.METHODS: The cDNAs were obtained from the spleen cells of BALB/c mice and the full length of mIL-1α gene was amplified by RT-PCR.Then the mIL-1α gene was inserted into a prokaryotic expression vector pET32a(+) and the resulting recombinant plasmid was transformed into E.coli BL21(DE3).After auto-induction,the mIL-1α protein was expressed and purified by electro-elution.An anti-mIL-1α polyclonal antibody was raised in New Zealand rabbits after immunization with the purified mIL-1α and the titer was determined by ELISA.The specificity of the polyclonal antibody was identified by Western blot and flow cytometry.RESULTS: The recombinant prokaryotic expression vector pET32a(+)-IL-1α was successfully constructed,and the mIL-1α protein was expressed and purified.ELISA showed the titer of the anti-mIL-1α serum was 1∶25 600.Western blot and flow cytometry demonstrated the high specificity of the polyclonal antibody to IL-1α.CONCLUSION: The rabbit anti-mIL-1α polyclonal antibody with high titer and specificity has been prepared after immunization with the purified mIL-1α protein,facilitating further functional studies of IL-1α.