|
|
|
| Cloning , Expression of Duck Interleukin-18 Mature Protein Gene and Biological Activity |
| |
| |
|
|
|
|
Abstract One pair of primers was designed and synthesized according to duck IL-18 gene sequences published in GenBank．Duck IL-18 mature protein gene was amplified by RT-PCR from total RNA extracted directionally from ma duck splenocyte ,then was cloned and sequenced．The result suggested that the nucleotide sequence of duck IL-18 mature protein gene be consisted of 513 bp in length, encoding 170 amino acid residues．A prokaryotic expression plasmid of mDuIL-18 , pQE30-mDuIL18 , was obtained by subcloning the encoding region of the DuIL-18 mature peptide into pQE30. pQE30-mDuIL18 was transformed E.coli M15 ,and induced by IPTG. The expression of p IL-18 mature protein gene was identified by SDS-PAGE and Western-blotting. The results revealed it had a molecular weight of 19755,and could be specifically recognized by the rabbit sera to chicken IL-18. The expressed products exist at the form of inclusion body. After being degenerated and then renaturated, the activity of the inclusion bodies were detected by by MTT. In ducks injected intramuscularly with rduIL-18 protein (150 ng or 200 ng per duck, respectively) and AIV vaccine 2 weeks after immunization, the average titers of HI antibodies to AIV reached 7.5–7.7 log 2, while the average titers of HI antibody to AIV were 6.3–6.6 log 2 in ducks only vaccinated with AIV vaccine or with 100 ng rduIL-18 and AIV vaccine. The results clearly showed that 150 ng rduIL-18/duck strengthened in vivo immune responses induced by the inactivated oil emulsion AIV vaccine.Development of recombinant pQE30-mDuIL18 paved the way for future study of biological function of expressed product and development of recombinant fowlpox viruses(rFPV) coexpressing duck IL-18 and protective antigen gene.
|
|
Received: 09 October 2006
|
|
|
|
|
|
|
