Prokaryotic expression and polyclonal antibody preparation of RelB protein in golden pompano(Trachinotus ovatus)

【Objective】The polyclonal antibody against Trachinotus ovatus RelB protein(TroRelB) was prepared tolay the foundation for further study on the structure, function and protein-protein interaction of TroRelB protein.【Method】The recombinant plasmid pET-32a-TroRelB was constructed by linking TroRelB gene to expression vector pET-32a then transformed into Escherichia coli BL21(DE3) competent cells.The protein expression was induced by isopropyl-β-D-thiogalactoside(IPTG).The recombinant TroRelB protein purified with a Ni-IDA agarose resin column was applied to immunize Balb/C mice to prepare polyclonal antibody.The titer and specificity of this antibody were detected by ELISA and Western blotting assay respectively.【Result】The BL21(DE3) competent cells were transformed by recombinant plasmid pET-32a-TroRelB and the recombinant TroRel Bprotein with a molecular weight of 84.0 kD was successfully expressed mainly in the form of inclusion body by IPTG inducing.The concentration of the recombinant TrorelB protein which could be recognized by antibody of anti-His tag was 0.498 mg/mL after purification by Ni-IDA agarose resin column.The anti-TrorelB serum(polyclonal antibody) was obtained by immunizing Balb/Cmice with the purified recombinant TrorelB protein.The ELISA result showed that the titer of this polyclonal antibody was 1:256000, and the Western blotting result showed that both 5 and 15 ng of the recombinant TrorelB protein reacted specifically withthe polyclonal antibody while the negative control(serum before the immune) did not show the expected band.【Conclusion】The recombinant TrorelB protein induced by the prokaryotic system is mainly expressed in the form of inclusion body and carries His label.The polyclonal antibody of TrorelB prepared by immunizing Balb/C mice with this recombinant protein possess strong specificity and high antibody titer, which can be used to further study the biological function of TrorelB protein and reveal the immune mechanism of T.ovatus.