Purification And Activities Investigation Of Tentacle-only Extract From The Jellyfish Cyanea Capillata

INTRODUCTIONWith the climate warming and offshore eutrophication, jellyfish outbreaks have become increasingly frequent over global ocean. Poisonous jellyfish stings are becoming the most common marine biological injuries. Research shows that jellyfish toxins are a group of novel peptide toxins, with the relative molecular weight ranging from 10-600 kDa. Jellyfish toxins possess a variety of biological activities, such as cardiovascular toxicity, hemolysis toxicity, neuromuscular toxicity, skin toxicity, and so on, of which the cardiovascular toxicity is one of the main toxicities and considered to be the major reason to induce death.In the recent years, many papers reported the cardiovascular toxicity of jellyfish toxins, but most of them still took the crude venom as the sample. Up to now, there is no report which has successfully purified and identified the protein with cardiovascular toxicity. It is related to the own characteristics of jellyfish toxins. The toxins are instability, easy to breakdown and lose its activities in the purification process. Purification and identification of jellyfish cardiovascular protein has become one of the major difficulties in studies on jellyfish toxins.In this thesis, the jellyfish Cyanea capillata, one kind of the major poisonous jellyfish in the southest coast of China, was taken as the research object. In the first part, I studied the methods of preparation the tentacle-only extract (TOE) and nematocyst venom (NV) from the tentacles of C. capillata, to choose a suitable sample for purification of cardiovascular toxic protein. In the second part, I tested the toxicity of the selected samples to determine the dose for active monitoring of each component during the purification. In addition, I also detected the activities of the samples which dialysised against common buffers, in order to get the best purification experiment buffer. In the third part, I pretreated the sample by salting out and ultrafiltration at first and purifiered the sample with ion exchange chromatography, gel filtration chromatography and other methods of chromatography. The toxicity of each component was monitored by cardiovascular toxicity testing models and the composition of each component was determained by SDS-PAGE; In the final part, we determined the primary structure of the active protein by a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), studied their sequences characteristics and analysised their possible biological activities.METHODSPartâ… : Extraction of tentacle-only extract and nematocyst venom TOE and nematocysts were separated by autolysis, filtration and centrifugation. Influences of different autolysis times on the autolysis rate of tentacles and the acquisition of nematocysts were observed. The types of nematocysts from C. capillata were observed by optical microscopy. The effects of repeated freeze-thawing, glass homogenized, mini-bead beater and others methods?on the preparation of nematocysts venom were also observed. The cardiovascular and haemolytic activities of both TOE and NV were determined parallelly. SDS-PAGE was used to analyze the protein composition of the two different samples and chose purification sample after comprehensive evaluationPartâ…¡: Toxicity studies of tentacle-only extractThe experiment was conducted on the LD50 of TOE in Kunming mice by tail vein injection (ml of toxin protein to per 10 g body weight): Total 128 Kunming mice (half male and half female) were used to measure 100% lethal dose (Dm) and the maximum safe dose (Dn) first, and the rest mice were randomly divided in to nine groups to determine the LD50 of TOE using improved karber's method. The feeding, behavior, excretion, poisoning and death situation were observed and recorded for seven days. External jugular vein cannulation was utilized for TOE administration with different concentrations while femoral artery cannulation for blood pressure monitoring. The blood pressure changes could reflect the TOE cardiovascular toxicity, thus completing the preliminary determination of the critical lethal value Dt. Dt can provide dose guidance for activity determination of the following purification components. TOE was immersed in several common anion or cation exchange buffer solutions overnight dialysis to assay the influence on the lethal toxicity of TOE. Partâ…¢: Purification and identification of tentacle-only extractFirst of all, I used ammonium sulfate precipitation and ultrafiltration methods to pretreat TOE, in order to remove a large number of miscellaneous proteins and obtain the possible molecular weight range of target protein. Using DuoFlow (Bio-Rad Company) and ?KTA Purifier (GE Healthcare) protein purification system as the main technology platform combined with gel filtering and ion exchange chromatography to separate and purify C. capillata TOE crude toxin. External jugular vein cannulation was utilized for TOE administration with different concentrations while femoral artery cannulation for blood pressure monitoring. The blood pressure changes could reflect the TOE cardiovascular toxicity, and SDS-PAGE was used to analyze the protein composition. We determined the primary structure of active protein by a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), studied their sequences characteristics and analysised their possible biological activities.RESULTSPartâ… : Partâ… : Preparation of tentacle-only extract and nematocyst venomTOE and nematocysts from the jellyfish C. capillata were successfully separated by autolysis, filtration and centrifugation. Different autolysis times can affect the autolysis rate of tentacles and the acquisition of nematocysts. At 0 h group, the autolysis accomplished with the help of sonication, and at the 2 h group, the autolysis accomplished with the help of glass rod. The undissolved tentacles of these two groups are lower than the 4 - 48 h groups, and the acquisition of nematocysts are more than other groups. There are little undissolved tentacles at the groups of 48 h, 96 h (m / v, 1:1), 96 h (m / v, 1:2) and 96 h (m / v, 1:3), and the 96 h (m/v, 1:1) group get most quality of nematocysts. In a word, the optimum condition for C. capillata tentacles autolysis was that the tentacles autolysis for 96 h at the rate of 1:1 (tentacles mass: the volume of sterile sea water). The microscopic view of the C. capillata nematocyst showed that it? consists of two parts: cyst and ecthoraeum. The nematocysts can be divided into banana-shaped and ellipse-shaped from shape, and mainly in banana-shaped. The glass homogenate machine could be used to repture the nernatocysts of jellyfish C. capillata and to extract the nernatocyst venom. Cardiovascular and haemolytic activities of TOE and NV were determined parallelly. The results showed that both of them have strong cardiovascular and haemolytic activities. The concentration-associated haemolytic curves were both"S"-shaped. Cardiovascular activities of TOE were significantly stronger than that of the same dose of NV. SDS-PAGE showed that protein bands of TOE owed maximum protein numbers, mainly concentrated in the large molecular weight region; protein bands of NV distributed more evenly with richer at the smaller weight regions.Partâ…¡: Toxicity studies of tentacle-only extractThe LD50 of TOE is 4.25 mg/kg (mg of toxin protein to per kg body weight) in Kunming mice by tail vein injection, and toxic symptoms of the mice are obviously dependent on the TOE doses. At the lower doses (less than 5 mg/kg i.v.), the mice showed progressive malaise, slowness, crouching and weakness, but little change in breathing. The deaths mainly occurred within 48 h, and if did not die within 48 h, the mice will return to normal gradually. Autopsy clearly showed the heart enlargement and edema, and the lung edema and congestion with bright red surface. At the intermediate doses (5-10 mg/kg i.v.), deep and fast breath was showed in the early, and then breathing was weakened gradually and arrested finally. Additionally, progressive malaise, slowness, crouching, muscle trembling and some double leg twitching were also observed, and most mice died within 2 h. At the higher doses (more than 10 mg/kg i.v.), deep and fast breath emerged rapidly, and the mice also showed rapidly progressive whole body trembling, double leg twitching, convulsion, opisthotonos and death. Most mice died within 5 min, and all the mice died within 15 min. However, both the heart and lung autopsy are almost normal.The critical lethal value Dt of TOE is 0.8 mg/kg (mg of toxin protein to per kg body weight) in SD rats. Intravenous injections within TOE will quickly (10-30 s) introduce a mild and transient rise response of blood pressure, and then accompanied by a drastic drop. At the 0.7 mg/kg group, blood pressure of the rats had a significant drop during 1 to 2 min, and the blood pressure value reached the minimum (about 40 mmHg). Over the time, blood pressure recovered gradually, and reached stability value at 80 mmHg after 30 min. At the 0.8 mg/kg group, blood pressure of the rats had a significant drop during 0.5 to 2 min, and the blood pressure value continued to decline after 2 min later. Continuous observation revealed that the blood pressure in some rats had gradually returned to 70 mmHg, but the blood pressure in other rats could not recovere and died after 20 min. At the 0.9 mg/kg group, blood pressure of the rats decreased significantly during 1 to 2 min, accompanied by violent convulsions in rats. Blood pressure continued to fall and all the rats died in 20 min to 40 min. All these can be concluded that the critical lethal value Dt of TOE is 0.8 mg/kg in SD rats.Partâ…¢: Purification and identification of tentacle-only extractPretreated TOE with ammonium sulfate precipitation and ultrafiltration methods?could initially identified that the components about 27 kDa may include the cardiovascular toxicity protein of TOE. Then the candidate components were purifiered by gel filtering and ion exchange chromatography, and a 27 kDa protein was found to be the target cardiovascular protein.CONCLUSIONTOE from jellyfish C. capillata has significantly stronger cardiovascular toxicity than that of NV at the same doses. Compared with NV, the preparation of TOE is simple and easy to operate, but the quality of TOE is much larger than the NV.?Therefore, TOE is the most suitable sample for separation and purification of cardiovascular proteins.TOE has strong toxicities. Its LD50 is 4.25 mg/kg in Kunming mice by tail vein injection, and its critical lethal value Dt is 0.8 mg/kg in SD rats.Using different chromatography methods, combined with cardiovascular toxicity monitoring and SDS-PAGE analysis, a 27 kDa protein is found to be the target cardiovascular protein.