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A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.