Cloning, expression and antioxidant activity of a novel collagen from Pelodiscus sinensis.

Collagen is the main structural protein of various connective tissues in animals and naturally plays an important role within the body. It is increasingly used within certain areas, such as medicine, citology and cosmetology. The soft-shelled turtle (Pelodiscus sinensis) is a commercially important aquatic species rich in collagen. In this study, a novel collagen gene fragment of 756 bp, which encodes 252 deduced amino acid residues, including 25 conserved Gly-X-Y motifs, was cloned from a soft-shelled turtle. Recombinant soft-shelled turtle collagen (rSTC) was stably expressed in Escherichia coli Rosetta and purified by His GraviTrap affinity columns. The antioxidant activities of rSTC were measured using hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. The results showed that rSTC quenched the free radicals in a dose-dependent manner. The hydroxyl radical scavenging activity (HRSA) of rSTC was 98.9 % at a concentration of 3 mg/mL. At a concentration of 5 mg/mL, rSTC exhibited a DPPH radical scavenging activity of 32.7 %. At the tested concentrations, rSTC exhibited higher HRSA and lower DPPH radical scavenging activity.

Rapid immunochromatographic test strip to detect swimming crab Portunus trituberculatus reovirus.

Swimming crab reovirus (SCRV) is the causative agent of a serious disease with high mortality in cultured Portunus trituberculatus. A rapid immunochromatographic assay (ICA) was developed in a competitive assay format and optimized for the detection of SCRV. The gold probe-based ICA test comprised SCRV antigen and goat anti-chicken egg yolk antibody (IgY) sprayed onto a nitrocellulose membrane as the test line and control line, respectively. IgY-gold complexes were deposited onto the conjugate pad as detector reagents. The method showed high specificity with no cross-reactivity with other related aquatic pathogens. The detection limit of the ICA strip was 50 µg ml⁻¹. To evaluate the performance of the ICA test, the strip and an enzyme-linked immunosorbent assay (ELISA) were applied to the same samples (n = 90 crabs). The strip successfully detected SCRV in all of the artificially infected samples. Furthermore, the ICA strip and ELISA tests had high consistency (98.28%). The strip assay requires no instruments and has a detection time of less than 10 min. It is portable and easy to perform in the field. These results indicated that the developed strip could be a promising on-site tool for screening pooled crabs to confirm SCRV infection or disease outbreaks.

Development of a colloidal gold immunochromatographic strip for the rapid detection of soft-shelled turtle systemic septicemia spherical virus.

A colloidal gold immunochromatographic strip (ICS) test based on a competitive format was developed for the rapid detection of soft-shelled turtle systemic septicemia spherical virus (STSSSV) in turtle and fecal samples. Specific egg yolk antibodies (IgY) against STSSSV were labeled with colloidal gold and used as probes in the one-step test strip. Antigen (STSSSV) and goat anti-chicken IgY were drawn on the nitrocellulose membrane as the test line and control line, respectively. When STSSSV standard samples (0-100µg/mL) were detected by the strips, the visual limit of detection (LOD) was found to be 50.0µg/mL. The ICS test showed high stability; the strips were stable for at least 3 months at 4°C without significant loss of activity. There was no obvious cross-reactivity with other aquatic pathogens. The assay can be performed within 5-10min. Analysis of STSSSV in turtle samples revealed that data obtained from the ICS test were in a good agreement with those obtained by ELISA. The positive results of fecal samples suggested that this method could be used to detect STSSSV while protecting the animals' welfare. The ICS assay does not need specialized equipment or a technician and can be used as a reliable, rapid, cost-effective and convenient qualitative tool for on-site diagnosis.