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

Published online by Cambridge University Press:  13 February 2008

Chen Hong-Ying ,
Cui Bao-An ,
Xia Ping-An ,
Li Xin-Sheng ,
Yang Ming-Fan  and
Zheng Lan-Lan
Chen Hong-Ying
Affiliation:
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China Animal Food Safety Key Laboratory, Henan Province, Zhengzhou 450002, China
Cui Bao-An*
Affiliation:
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China Animal Food Safety Key Laboratory, Henan Province, Zhengzhou 450002, China
Xia Ping-An
Affiliation:
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China
Li Xin-Sheng
Affiliation:
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China
Yang Ming-Fan
Affiliation:
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China Animal Food Safety Key Laboratory, Henan Province, Zhengzhou 450002, China
Zheng Lan-Lan
Affiliation:
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China Animal Food Safety Key Laboratory, Henan Province, Zhengzhou 450002, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

Duck 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.

Keywords

biological activitycloningduck interleukin-18expressionmature protein gene
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 4 , Issue 3 , December 2007 , pp. 207 - 212
Copyright
Copyright © China Agricultural University and Cambridge University Press 2007

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Footnotes

First published in Journal of Agricultural Biotechnology 2007, 15(4): 584–588

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