Language：English   Publisher：Protein Expression and Purification  

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4–2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

DOI： 10.1016/j.pep.2024.106450

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Language：English   Publisher：Human Antibodies  

BACKGROUND: A cost-effective and eco-friendly method is needed for the assessment of humoral immunity against SARS-CoV-2 in large populations. OBJECTIVE: We investigated the performance of an ELISA that uses silkworm-produced proteins to quantify the strain-specific anti-Spike IgG (anti-S IgG) titer. METHODS: The OD values for the anti-His-tag antibody, a standard material of ELISA quantification, were measured. Correlations between the ELISA for each strain and the Abbott SARS-CoV-2 IgG II Quant assay for the wild type were evaluated with serum samples from nine participants with various infection and vaccination statuses. RESULTS: Linear dose-responses were confirmed by high coefficients of determination: 0.994, 0.994, and 0.996 for the wild-type, Delta, and Omicron (BA.1) strain assays, respectively. The coefficient of determination for the wild-type and Delta strain assays was high at 0.959 and 0.892, respectively, while the Omicron strain assay had a relatively low value of 0.563. Booster vaccinees showed similar or higher titers against all strains compared to infected persons without vaccination. The Omicron-infected persons without vaccination had lower antibody titers against wild type than did the vaccinated persons. CONCLUSIONS: This study provides data indicating that the ELISA with silkworm-produced proteins makes it possible to discriminate and quantify the strain-specific anti-S IgG antibody induced by vaccination or infection.

DOI： 10.3233/HAB-230006

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Language：English   Publisher：Insects  

Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. In this study, we attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances our understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies.

DOI： 10.3390/insects14080684

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Language：English   Publisher：Insect Biochemistry and Molecular Biology  

O-glycosylation of secreted and membrane-bound proteins is an important post-translational modification that affects recognition of cell surface receptors, protein folding, and stability. However, despite the importance of O-linked glycans, their biological functions have not yet been fully elucidated and the synthetic pathway of O-glycosylation has not been investigated in detail, especially in the silkworm. In this study, we aimed to investigate O-glycosylation in silkworms by analyzing the overall structural profiles of mucin-type O-glycans using LC–MS. We found GalNAc or GlcNAc monosaccharide and core 1 disaccharide (Galβ1-3-GalNAcα1-Ser/Thr) were major components of the O-glycan attached to secreted proteins produced in silkworms. Furthermore, we characterized the 1 b1,3-galactosyltransferase (T-synthase) required for synthesis of the core 1 structure, common to many animals. Five transcriptional variants and four protein isoforms were identified in silkworms, and the biological functions of these isoforms were investigated. We found that BmT-synthase isoforms 1 and 2 were localized in the Golgi apparatus in cultured BmN4 cells and functioned both in cultured cells and silkworms. Additionally, a specific functional domain of T-synthase, called the stem domain, was found to be essential for activity and is presumed to be needed for dimer formation and galactosyltransferase activity. Altogether, our results elucidated the O-glycan profile and function of T-synthase in the silkworm. Our findings allow the practical comprehension of O-glycosylation required for employing silkworms as a productive expression system.

DOI： 10.1016/j.ibmb.2023.103936

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Language：English   Publisher：Vaccine  

Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9 mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40 nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS.

DOI： 10.1016/j.vaccine.2022.12.015

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Event date： 2024.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：農林水産技術会議事務局筑波産学連携支援センター   Country：Japan  

Event date： 2024.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：農林水産技術会議事務局筑波産学連携支援センター   Country：Japan  

Event date： 2024.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：農林水産技術会議事務局筑波産学連携支援センター   Country：Japan  

Event date： 2024.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：農林水産技術会議事務局筑波産学連携支援センター   Country：Japan