Abstract
Neosporosis, which is caused by Neospora caninum, is a well-known disease in the veterinary field. Infections in pregnant cattle lead to abortion via transplacental (congenitally from mother to fetus) transmission. In this study, a N. caninum profilin (NcPROF), was expressed in silkworm larvae by recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid and was purified from the hemolymph. Three NcPROF constructs were investigated, native NcPROF fused with an N-terminal PA tag (PA-NcPROF), PA-NcPROF fused with the signal sequence of bombyxin from B. mori (bx-PA-NcPROF), and bx-PA-NcPROF with additional C-terminal transmembrane and cytoplasmic domains of GP64 from BmNPV (bx-PA-NcPROF-GP64TM). All recombinant proteins were observed extra- and intracellularly in cultured Bm5 cells and silkworm larvae. The bx-PA-NcPROF-GP64TM was partly abnormally secreted, even though it has the transmembrane domain, and only it was pelleted by ultracentrifugation, but PA-NcPROF and bx-PA-NcPROF were not. Additionally, bx-PA-NcPROF-GP64TM was successfully purified from silkworm hemolymph by anti-PA agarose beads while PA-NcPROF and bx-PA-NcPROF were not. The purified bx-PA-NcPROF-GP64TM protein bound to its receptor, mouse Toll-like receptor 11 (TLR-11), and formed unique nanoparticles. These results suggest that profilin fused with GP64TM was secreted as a nanoparticle with binding affinity to its receptor and this nanoparticle formation is advantageous for the development of vaccines to N. caninum.
Highlights
Neosporosis is a disease caused by apicomplexan parasites such as Neospora caninum and Neospora hugheshi [1]
To evaluate the expression and purification of fusion native N. caninum profilin (NcPROF) fused with an N-terminal PA tag, fused with the signal sequence of bombyxin from B. mori fused with the transmembrane and cytoplasmic domains of GP64 from Bombyx mori nucleopolyhedrovirus (BmNPV), NcPROF was expressed in two other NcPROF constructs, one fused with a PA tag (PA-NcPROF) and PA-NcPROF fused with the signal sequence of bombyxin from B. mori
The GP64TM were used as a fusion partner with NcPROF as a target protein to be incorporated into the cell membrane [19]
Summary
Neosporosis is a disease caused by apicomplexan parasites such as Neospora caninum and Neospora hugheshi [1]. Most of the specific antigen proteins are secreted from each organelle and are located at the apical end of the N. caninum parasite and have been extensively studied as recombinant vaccine candidates against N. caninum infection [7,8]. We demonstrated the capability of baculoviruses displaying N. caninum-derived antigens, such as surface antigen 1 (NcSAG1), SAG1-related sequence 2 (NcSRS2) and microneme protein 3, as an alternative method to control neosporosis because the combination of three antigens could induce T-cell activation and interferon gamma (IFN-γ) production and suppress N. caninum infection in mice [9]. Profilin is known as a small actin-binding protein located at the apical end of N. caninum tachyzoites and is essential for invasion of the host cell by regulating the polymerization and depolymerization of actin filaments [11]. The bx-PA-NcPROF-GP64TM was successfully purified from silkworm hemolymph and the binding of bx-PA-NcPROF-GP64TM with recombinant mouse TLR11 (mTLR11), and the morphological analysis of the nanoparticles were reported
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