Secretory Nanoparticles of Neospora caninum Profilin-Fused with the Transmembrane Domain of GP64 from Silkworm Hemolymph

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.

Ni-modified magnetic nanoparticles for affinity purification of His-tagged proteins from the complex matrix of the silkworm fat body

“…Both plasmids were constructed and then utilized for the generation of a recombinant BmNPV bacmid. Subsequently, the recombinant baculovirus was generated in cultured Bm5 cells according to our previous reports [ 31 ]. The cell culture supernatant was collected and used for serial infections to obtain high-titer virus stocks, which were employed to infect silkworm larvae.…”

Humoral Immune Response in a Mouse Model Induced With Dengue Virus-like Particles Serotypes 1 and 4 Produced in Silkworm Larvae

“…Each recombinant BmNPV bacmid was combined with chitosan (Sigma-Aldrich, Tokyo, Japan) and administered to fth instar silkworm larvae (Ehimesansyu, Ehime, Japan). The hemolymph containing recombinant BmNPV was collected from the larvae 6-7 days after injection (dpi) and mixed with 1-phenyl-2-thiourea at 1 mM, with aliquots of hemolymph stored at -80 °C before use (Boonyakida et al 2021;Suhaimi et al 2019;Utomo et al 2019;Utomo et al 2020).…”

Ni-modified Magnetic Nanoparticles for Affinity Purification of His-tagged Proteins From the Complex Matrix of the Silkworm Fat Body

“…Both plasmids constructed and then utilized for the generation of a recombinant BmNPV bacmid. Subsequently, the recombinant baculovirus was generated in cultured Bm5 cells according to our previous reports [25]. The cell culture supernatant was collected and used for serial infections to obtain high-titer virus stocks, which were employed to infect silkworm larvae.…”