Abstract
Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.
Highlights
Due to enhanced anthropogenic activities, cadmium (Cd) contamination of the aquatic environment has increased during recent years [1], [2], and is frequently found in the Yellow River and Yangtze River of China [3], [4]
A single nucleic acid band was detected in lane 2 with a molecular mass of 3300 bp, and another two nucleic acid bands were detected in lane 1, respectively with a molecular mass of 3000 bp and 200bp, which were substantially consistent with the theoretical molecular mass of the phoA and MT, respectively
MT extract was fractionated by Sodium dodecyl sulfate (SDS)-PAGE, and protein bands were detected with a molecular mass of 7 kDa
Summary
Due to enhanced anthropogenic activities, cadmium (Cd) contamination of the aquatic environment has increased during recent years [1], [2], and is frequently found in the Yellow River and Yangtze River of China [3], [4]. Crustaceans are considered as suitable indicator species indicating environmental pollution in aquatic ecosystems because of their sediment-associated and less mobile life habits. Some crustacean species, such as the crayfish Astacus leptodactylus [19] and the crab Carcinus maenas [20] were chosen for the study of aquatic contamination. The freshwater crab Sinopotamon yangtsekiense became the subject of our molecular approaches This crab species is widely distributed in the Yangtze, the Huaihe River drainage basin, and the Yellow River Valley of China. We purified the MT of S. yangtsekiense through recombinant MT, exploiting the MT antiserum, and developed an ELISA to assess MT content in S. yangtsekiense and to monitor environmental Cd pollution
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