title: Expression of biologically active murine interleukin-18 in Lactococcus lactis.
abstract: The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3-4 μg mL-1and 0.6-0.7 ng mL-1, respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces.
authors.affiliation: Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Sadegh
authors.initials: S
authors.lastname: Feizollahzadeh
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Khanahmad
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Ilnaz
authors.initials: I
authors.lastname: Rahimmanesh
authors.affiliation: Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Mazdak
authors.initials: M
authors.lastname: Ganjalikhani-Hakemi
authors.affiliation: Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Andalib
authors.affiliation: Acquired Immunodeficiency Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Sanei
authors.affiliation: Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran rezaei@mui.ac.ir.
authors.forename: Abbas
authors.initials: A
authors.lastname: Rezaei
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007371:Interferon-gamma / Q000096:biosynthesis*; D020382:Interleukin-18 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D009092:Mucous Membrane / Q000276:immunology* / Q000382:microbiology; D009561:Nisin / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D020382:Interleukin-18; D011994:Recombinant Proteins; D009561:Nisin; D007371:Interferon-gamma; C102526:nisin A
keywords: Lactococcus lactis; T cells; biological assay; cloning; expression; interleukin-18