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.
Subject(s)
Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay/veterinary , Goats/immunology , Interleukin-18/analysis , Interleukin-18/immunology , Animals , Base Sequence , Cattle , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay/methods , Female , Fluorescent Antibody Technique, Indirect/methods , Fluorescent Antibody Technique, Indirect/veterinary , HEK293 Cells , Humans , In Vitro Techniques , Interleukin-18/genetics , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Species SpecificityABSTRACT
AIM: To study the technique of boosting the renaturation yield of rChIL-18 by using artificial molecular chaperone composed of cetyl trimethyl ammonium bromide (CTAB) and beta-cyclodextrin(beta-CD). METHODS: The recombinant plasmid of mChIL-18 prokaryotic expression was transformed into E.coli BL21 (DE3) strain and then induced by IPTG at 37 degrees Celsius. The recombinant mChIL-18 was expressed efficiently in inclusion bodies in E.coli. After crushed and washed, the inclusion bodies were thoroughly denatured with 6 mol/L of guanidine hydrochloride, and then the artificial molecular chaperone was used to promote protein refolding. After the rehabilitation of products was purified by bag filter, its activity was detected by lymphocyte proliferation assays. RESULTS: The SDS-PAGE analysis indicated the expressed ChIL-18 protein had molecular weight of 44000. The expressed product existed in the form of inclusion body.Two protein bands of Mr 44000 and 26000 appeared on SDS-PAGE gel. The percentage of renaturation was 42.54 with artificial molecular chaperone.The results of MTT assay showed the expression of ChIL-18 protein in E.coli BL21 (DE3) greatly induced the proliferation of chicken T lymphocytes. CONCLUSION: The artificial chaperone technique can obviously boost the renaturation yield of rChIL-18. The purified and expressed product of fusion chicken Interleukin-18 gene in E.coli have relativity high bioactivity.
