Transient Co-Expression of Bioactive Murine Interferon-Gamma and HBsAg in  Tobacco and Lettuce Leaves

Sara  Mohammadzadeh; Mahshid  Amiri; Parastoo  Ehsani

doi:10.18502/ajmb.v15i4.13493

Sara Mohammadzadeh Department of Medical Biotechnology, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
Mahshid Amiri Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD, United States
Parastoo Ehsani Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v15i4.13493

Keywords: Agrobacterium, Cytokines, Hepatitis B surface antigens, Plant proteins

Abstract

Background: The synchronous expression of antigen and adjuvant proteins in plant hosts presents an intriguing potential for vaccine production and the enhancement of appropriate immune responses. In this study, we examined the expression of bioactive murine interferon-gamma (mIFN-γ) along with HBsAg in tobacco and lettuce leaves aimed to further perform the analysis of immune responses in the mouse model.

Methods: Monocistronic and bicistronic cassettes, carrying genes encoding mIFN-γ and HBsAg in various orders, were constructed. These cassettes were placed under the control of the 35S CaMV promoter and included the 5ʹ leader sequence of Tobacco Ech Virus (TEV). Through Agrobacterium infiltration, the cassettes were transferred into plant leaves. The concentration of mIFN-γ in different constructs and HBsAg was tested by ELISA. Murine IFN-γ was characterized through Western blotting, and its bioactivity was evaluated by assessing the up-regulation of MHC class II in macrophages derived from mouse bone marrow.

Results: Extracts of agroinfiltrated leaves contained recombinant mIFN-γ and HBsAg proteins at about 14 unit/mg and 50 ng/mg of soluble protein, respectively. Subsequently, mIFN-γ was purified from the plant extract and its ability to up-regulate MHC class II in mouse bone marrow-derived macrophages was confirmed by immunofluorescence.

Conclusion: The co-expression of recombinant HBsAg and mIFN-γ using TEV 5ʹ leader-based cassettes in tobacco and lettuce leaves produced both proteins with active mIFN-γ in different concentrations. The attractive utility and feasibility of using plant transient co-expression systems aimed to co-delivery of vaccine antigen and appropriate cytokine to elicit immune response for different applications.