Cloning and expression of Ma duck interleukin-18 mature protein gene and biological activity detection

Abstract

AbstractDuck interleukin (IL)-18 mature protein gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from Ma duck (Tadorna ferruginea) splenocytes. The PCR product was cloned into pGEM-T Easy vector for sequencing. The result revealed that the nucleotide sequence of duck IL-18 mature protein gene (mDuIL-18) consisted of a 513 bp band. A prokaryotic plasmid of mDuIL-18, pQE30-mDuIL18, was obtained by subcloning the encoding region of the DuIL-18 mature peptide into pQE30. pQE30-mDuIL18 transformed Escherichia coli M15. The expression of mDuIL-18 gene was identified by SDS-PAGE and Western blotting. Its molecular weight was 19.76 kDa, and could be specifically recognized by rabbit sera to chicken IL-18. The expressed products existed as inclusion bodies. After being degenerated, then renatured, the activities of the inclusion bodies were detected by methyl thiazolyl tetrazolium (MTT) assay. In ducks injected intramuscularly with mDuIL-18 protein (150 ng or 200 ng per duck) and Avian influenza virus (AIV) vaccine 2 weeks after immunization, the average titres of haemagglutination inhibition (HI) antibodies to AIV reached 7.5–7.7 log2, while those of HI antibody ranged between 6.3 and 6.6 log2 in ducks vaccinated with AIV vaccine only or with 100 ng mDuIL-18 and AIV vaccine. The results clearly showed that 150 ng mDuIL-18 per duck strengthened the in vivo immune responses induced by the inactivated oil emulsion AIV vaccine.