Kudoa septempunctata, a myxozoan, has been identified as the causative agent of foodborne illnesses associated with the consumption of raw olive flounder. To develop effective control methods against this parasite, fundamental research—including viability determination, transcriptome analysis, and antigenicity assessment of K. septempunctata myxospores—is required. This research necessitates the purification of the parasites. Sequential trypsin digestion, followed by density gradient purification, was performed to isolate the K. septempunctata myxospores. Further purification was achieved through fluorescence-activated cell sorting at a concentration of 106 to 107 myxospores/ml. The results demonstrated that the combination of trypsin digestion and density gradient methods consistently produced approximately 40 times more viable myxospores than the density gradient method alone. Additionally, the fluorescence-activated cell sorting method enhanced the purity of the myxospores by approximately 10%. The procedures described in this study will support research (such as RNA-sequencing, proteomics, vaccine antigen preparation) aimed at developing control methods for K. septempunctata including the fundamental research.
In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited 42-57% homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GDSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cockroach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.
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