Abstract
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 vBmswp26-egfp bacmid. The vBmswp26-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.
Highlights
Microsporidia are a large group of highly adapted obligate intracellular parasites, which can infect a wide range of vertebrate and invertebrate hosts, including insects, fish, crustaceans, and mammal groups [1]
Recent research suggested that the spore wall proteins (SWPs) are involved in the complex infection mechanism, and are responsible for adherence of the microsporidian spores to host cells, which is an integral part of activation and host cell infection [5]
A 685-bp DNA fragment encompassing the SWP26 gene of N. bombycis and a 736-bp DNA fragment encompassing the enhanced green fluorescent protein (EGFP) gene were synthesized by PCR using primer sets specific to the corresponding genes (Figure 1(a))
Summary
Microsporidia are a large group of highly adapted obligate intracellular parasites, which can infect a wide range of vertebrate and invertebrate hosts, including insects, fish, crustaceans, and mammal groups [1]. Most of the entomopathogenic microsporidia belong to the genus Nosema, with more than 150 described species from 12 insect orders [2,3]. The microsporidia first described in the literature, is a silkworm parasite that causes severe economic losses in the sericulture industry. Key to the protection of these spores is a thick spore wall composed of a double layer of a chitin-rich endospore and a proteinbased exospore [4]. Five spore wall proteins have been studied in N. bombycis: three exosporal proteins (SWP5, SWP26, and SWP32) and two endosporal protein (SWP25 and SWP30) [12,13,14,15,16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
