Search

Additional file 4: Figure S2. Cross-section of The Harvest. (a) The scanning electron microscopy (SEM) image shows the ground layer consisting of two layers under a paint layer. The upper ground layer contained lead white and calcium carbonate, and the bottom ground layer contained calcium sulfate only, or calcium sulfate and calcium carbonate. The sample was examined by SEM (JSM-5800LV; JEOL, Tokyo, Japan) equipped with an energy dispersive X-ray spectroscope (DX4; EDAX, Mahwah, NJ). (b) Schematic illustration of the cross-section.

Additional file 3: Figure S1. MRM chromatograms of marker peptides detected for location #2 of The Harvest. Detected marker peptides are indicated in boldface.

Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1–16) in the α1 chain. When purified collagen was digested, pQLSYGY (1–6) and pQLSYGYDEKSTG (1–12) were identified, while DEKSTG (7–12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH2 was digested, pQLSK(MOCAc)GYDEKSTG (1–12) and ISK(Dnp)P-NH2 (13−16) were readily identified, pQLSK(MOCAc)GY (1−6) and DEKSTGISK(Dnp)P-NH2 (7−16) were weakly detected, and DEKSTG (7–12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not. Pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. The former cleavage requires native collagen structure, while the latter cleavage does not.