Immunization balb/c mice against leishmania major through lactococcus lactis bearing sp15 antigen derived from phelebotomus papatasi
,1 Negar seyed
,2 Reza safaralizadeh
,3 Sima rafati
,4 Jesus valenzuela
,5 Tahereh taheri
1. Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
2. Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
3. Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
4. Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
5. Vector Molecular Biology Section, LMVR, National Institute of Allergy and Infectious Diseases, National Institutes of H
6. Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
Lactococcus lactis (l. lactis) as a nonpathogenic expression system can be used to delivery live vaccines. furthermore cytoplasm, proteins expression into extracellular or attached on the cell wall is possible, if the desired gene is cloned downstream of the relevant peptide signal. here, sp15 gene isolated from phlebotomus papatasi (sand fly vector for leishmania major (l. major)) alone or along with the enhanced green fluorescent protein (egfp) expressed on the cell-wall of l. lactis. then, the immunization potential of recombinant bacteria was evaluated against l. major on balb/c mice.
The codon optimized both sp15-egfp and egfp (as a control) genes were cloned in downstream of prtp signal peptide in pnz8121 vector and transformed into mc1061 as an intermediate host. the final constructs were electrotransformed into l. lactis (strain nz9000) and induced using the nice system. to confirm the cell-wall attached expressed proteins, membrane of bacteria were separated, precipitated and applied to analysis through western blotting and whole cell elisa methods using anti-gfp antibody. the several balb/c mice groups were immunized with different regimens. after challenging of immunized mice with l. major plus sgh, footpad swelling was measured weekly, and parasite load in lymph nodes were estimated using real-time pcr and limiting dilution at two different timepoint.
Both western blot and elisa analysis recognized a specific 42-kda band and high absorbance related to expressed sp15-egfp in membrane of bacterium. moreover, immunized mice with recombinant l. lactis-sp15-egfp have shown less swelling size in footpad and also lower parasite load in lns in comparison with immunized mice with wild-type bacteria or no vaccinated groups (p<0.05).
The expression of sp15-egfp on the cell-wall of l. lactis can proposed as a suitable candidate live vaccine for leishmaniasis.
Lactococcuslactis, leishmania major, sgh, sp15, vaccine.