Construction of eukaryotic express vector of duck interleukin 18 gene and identification of bioactivity of its expressed protein / 生物工程学报

ABSTRACT

Duck IL-18 gene was amplified from plasmid pGEM-DuIL-18 by PCR. The PCR product digested with Pst I and Xho I was inserted into eukaryotic express vector pcDNA3.1(+) to generate an recombinant expression plasmid pcDNA3.1/DuIL-18 (pDuIL-18), and transformed into Escherichia coli JM109. The recombinant colonies were identified by restriction enzyme digestion, PCR and sequencing. DNA sequence confirmed the correct sequence of the recombinant eukaryotic expression plasmid pDuIL-18 in the reading frame and the ligation part. After the transfection of pDuIL-18 into Cos7 cells, duck IL-18 mRNA was expressed in Cos7 cell. The SDS-PAGE analysis showed that the expressed duck IL-18 protein had molecular weight of 23 000 D. The results of methyl thiazolyl tetrazolium (MTT) assay showed that duck IL-18 protein expressed in Cos7 cell could induce significantly transformation of duck T lymphocytes. Immunoenhancement effect of recombinant expression plasmid pDuIL18 on avian influenza vaccine was observed by proliferation response of the T lymphocytes from spleen. It can obviously enhance the cell-mediated immune response.