23/08/1402
Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice
Sand fly salivary proteins have immunomodulatory and anti-inflammatory
features; hence, they are proven to perform important roles in the early
establishment of Leishmania parasite in the vertebrate host.
Among them, salivary apyrase with anti-hemostatic properties has a
crucial role during the blood meal process. In the present study, a
Genome-Walking method was used to characterize a full-length nucleotide
sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli
BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot
and ELISA results indicated that both recombinant apyrases could induce
antibodies in BALB/c. Moreover, a partial cross-reactivity between
Pkapy and Ppapy was found. In vitro stimulation of splenocytes from
immunized mice with the recombinant proteins indicated cross-reactive T
cell proliferative responses. Cytokine analysis revealed significant
production of IFN-γ (p < 0.001) and IL-10 (p < 0.01)
in response to Pkapy. In conclusion, the full-length nucleotide
sequence and molecular characteristics of Pkapy were identified for the
first time. Immunologic analyses indicated that Pkapy and Ppapy are
immunogenic in BALB/c mice and show partial cross-reactive responses.
The immunity to Pkapy was found to be a Th1-dominant response that
highlights its potential as a component for an anti-Leishmania vaccine.