Expression and bioactivity analysis of Acanthopagrus latus recombinant IL-1β

Abstract:

Interleukin 1β (IL-1β) gene was amplified from the total RNA of Acanthopagrus latus. The PCR product was subcloned into expression vector pQE30 and was subsequently transformed into E. coli M15 with IPTG inducement to be expressed. The recombinant protein was purified with Ni2+ chelating sepharose FF chromatography and the renaturation of the recombinant protein was subjected to gradient dialysis. The results of SDS PAGE and Western Blot analysis proved that the recombinant IL 1β with a molecular weight of 21kD was successfully expressed. The head kidney leucocytes of A. latus were isolated by using a discontinuous Percoll density gradient centrifugation and the density of 1.080g/ml was suitable for the isolation of head kidney leucocytes. The cultured head kidney leucocytes of the A. latus were stimulated by different final concentrations of rIL-1β and 5μg/ml LPS. The total RNA of the leucocytes was extracted after 4 h at 23℃. The RT PCR revealed that rIL 1β could up regulate the transcription of IL-1β at 20ng/ml and was equivalent to the effect of 5μg/ml LPS. The rIL-1β of A. latus showed efficacious bioactivity.