ABSTRACT
AIM: To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. METHODS: The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. RESULTS: The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. CONCLUSION: The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.
Subject(s)
Baculoviridae/genetics , Baculoviridae/metabolism , Interleukin-18/genetics , Interleukin-18/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Animals , Cell Line , Cell Proliferation/drug effects , Cloning, Molecular , Genetic Vectors/genetics , Genetic Vectors/metabolism , Goats , Insecta/cytology , Interferon-gamma/metabolism , Interleukin-18/pharmacology , Lymphocyte Activation/drug effects , Recombinant Proteins/pharmacology , Vesiculovirus/drug effectsABSTRACT
In order to develop a specific assay for the measurement of goat IL-18 level, two stable hybridoma cell lines were established which secreted IgG1 monoclonal antibodies (mAbs) against goat IL-18. Specific binding of two mAbs named 2E8 and 4C4 to recombinant goat IL-18 expressed in Escherichia coli was demonstrated in an ELISA and Western blotting. Results also showed that mAbs 2E8 and 4C4 bound to distinct epitopes in the ELISA additivity test. These two mAbs were applied in IFA analysis for the detection of goat IL-18 expressed in 293FT cells and in the sandwich ELISA for the measurement of goat IL-18 levels in LPS-stimulated PBMC. Results from this study demonstrated that mAbs against goat IL-18 recognize bovine and human IL-18 and could be used to measure IL-18 levels in different inflammations or immune responses in future studies.
