We successfully established a detection method which exhibited a markedly higher sensitivity than previously developed detection methods for Nosema bombycis by combining glass beads, FTA card, and LAMP. Spores of N. bombycis were first broken by acid-washed glass beads; the DNA was subsequently extracted and purified with the FTA card, and LAMP was performed using primers (LSU296) designed based on the sequence of the LSU rRNA of N. bombycis. The minimum detection concentration was 10 spores/mL. When this method was used to detect pebrine disease in silkworm egg, the detection rate for 500 silkworm eggs, in which only one egg was infected with N. bombycis, was 100 % under our optimized conditions. If the number of eggs in the sample increased to 800 or 1,000, the sample was divided into two equal portions, and the eggs were smashed with glass beads after the addition of 1 mL of TE buffer. The liquid in two tubes was later mixed and applied to the FTA card, and the detection rates were 100 %. Furthermore, the LAMP method established in our study could detect N. bombycis infection in silkworm 24 h earlier than microscopy.
The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this study, we used the Bac-to-Bac baculovirus expression system to express the spore wall protein SWP26, fused to enhanced green fluorescent protein (EGFP), in the silkworm BmN cell line. The SWP26 and EGFP genes were inserted into the baculovirus transfer vector pFastBac1. The transfer vector pFastBac1-swp26-egfp was transformed into the bacterium Escherichia coli DHl0Bac/Bombyx mori nucleopolyhedrovirus (BmNPV) to construct the recombinant vBm swp26-egfp bacmid. The vBm swp26-egfp bacmid DNA was then used to transfect BmN cells to obtain the recombinant baculovirus. Western blotting analysis of total protein lysates in BmN cells infected by the recombinant virus showed a protein band of approximately 51 kDa, which corresponded to the deduced molecular weight of the swp26-egfp fusion protein. In addition, a fluorescence signal was observed in the cytoplasm and nucleoplasm of transfected cells, indicating that SWP26 had been successfully expressed in BmN cells. The SWP26 expression system established in this study lays the foundation for additional molecular and cellular studies, especially those focused on the interaction between the SWP26 protein of Nosema bombycis and the proteins of the silkworm, Bombyx mori.
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