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Objective To obtain a single toxin component from the jellyfish Cyanea capillata and provide a foundation for the further study on bioactivity and function of the serine proteases from C.capillata.Methods Primers designed with restriction enzyme were used to amplify the coding region of cDNAs (CcSP1, CcSP2 and CcSP3).PCR fragments were ligated with the pET-24a( +) vector to construct the recombinant plasmids (pET24a-CcSP1, pET24a-CcSP2 and pET24a-CcSP3).After screening and identification,the recombinant plasmids were transformed into the Rosetta (DE3).plysS for protein expression.After induction with IPTG, SDS-PAGE and Western-blot were used to detect the expression of the recombinant proteins.Results SDS-PAGE showed that the proteins of rCcSP1, rCcSP2 and rCcSP3 were expressed in a single band at about 34 kDa, 42 kDa and 42kDa, respectively.Western-blot detection with anti-His antibody further confirmed that these recombinant proteins were His-tagged CcSP1, CcSP2 and CcSP3 fusion protein were obtained.Conclusion Prokaryotic recombinant plasmids of C.capillata serine proteases are contructed and recombinant proteins are obtained, which establishes the foundation for future study on the function of serine proteases from jellyfish.
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Objective To investigate the influencing factors of the protein stability and hemolytic activity of tentacle extract ( TE) from the jellyfish Cyanea capillata .Methods Effects of various factors and treatments on the protein stability and hemolytic ac-tivity of TE were explored by protein detection , hemolytic assay and SDS-PAGE analysis.Results TE caused a significant and dose-dependent hemolytic effect , and the HU50 of TE against 0.5%erythrocyte suspensions from SD rats was 226μg/ml.A 40℃water bath for 1 hour could effectively remove the contaminating proteins in TE .TE retained hemolytic activity at 4℃for 28 days but it was unsta-ble when kept at 25℃over 3 days.TE was active in the range from pH 6.0 to 11.0 and the optimum pH was 8.0.Various buffer solu-tions had significantly different effects on the stability and hemolytic activity of TE , and a good salting-out effect was observed on the hemolytic protein of TE while the concentration of ammonium sulfate solutions was greater than 26%.Conclusion A 40℃water bath for 1 hour could effectively remove the contaminating proteins in TE and reduce its viscosity .The optimum conditions for maintaining stability and hemolytic activity of TE were 4℃ and pH 8.0.The salting-out effect from 26% and more ammonium sulfate solutions would be conducive to the enrichment of hemolytic protein .
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Objective To investigate the potential role of the pore-formation in the hemolytic activity of tentacle-only extract (TOE) from the jellyfish Cyanea capillata. Methods The effects of various cations, including K+, Ca2+, Mg2+, Mn2+, Zn2+, Cu2+, Fe2+, La3+and NH4+ on the hemolytic activity of TOE were compared in two different test systems: 1% whole blood and 0. 45% erythrocyte suspension with the same erythrocyte concentration. Results The hemolytic activities of TOE in both tests were inhibited by Mn2+, Zn2+, La3+, Cu2+ and Fe2+, and were promoted to a minor extent by K+, Ca2+, Mg2+and NH4 +. The chelating agent EDTA also inhibited the hemolytic activity of TOE. Conclusion The pore-formation mechanism might play an important role in the hemolytic activity of TOE.
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Objective: To separate the nematocyst venom (NV) and tentacle-only extract (TOE) from the jellyfish Cyanea capillata and to analyze the difference between their haemolytic activities and the influencing factors. Methods: NV and TOE were separated by autolysis and centrifugation. The influence of concentration, temperature and pH value on the haemolytic activities of NV and TOE were observed. Results: NV and TOE were successfully separated. The concentration-associated haemolytic curves were "S" shaped for both NV and TOE. The HU50 of NV and TOE were 8 μg/ml and 67 μg/ml, respectively; and the haemolysis of NV was about 8.4 times of that of TOE. Temperature had great influence on haemolysis of both and the highest haemolysis were both at 40°C. pH value also had influence on haemolysis. The strongest haemolysis activity was found both at pH 8 and TOE was more sensitive. Conclusion: NV and TOE both have haemolysis activity, with the activity of NV stronger than that of the latter. The haemolysis activity is influenced by concentration, temperature and pH value.