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.