水母毒素的分离纯化及蛋白质组学研究
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摘要
水母(Jellyfish)分布广、种类多、资源丰富。水母毒素主要分布于触手的刺丝囊细胞中，是一类结构新颖且具有多种生物活性的蛋白。但是，由于水母种类的不同，其毒素的结构、生物活性也存在一定的差异。本论文以近年来经常在我国近海区域大规模出现的霞水母(Cyanea sp.)和沙蜇(Stomolophus meleagris)为研究对象，研究水母毒素的提取制备方法、生物活性、分离纯化及其蛋白质组学分析等，主要结果如下：
1.对水母毒素的提取制备方法进行了研究。以霞水母为研究对象，首先制备水母的刺丝囊细胞，然后采用组织研磨器Mini-beadbeater破碎刺丝囊细胞提取毒素，并且研究了提取过程中蛋白酶抑制剂对霞水母毒素溶血活性的影响，结果发现1mmol/LEDTA和4μg/mL PepstantinA能够有效地保护霞水母毒素的溶血活性。
2.研究了水母毒素的溶血活性，并经纯化得到具有溶血活性的蛋白。霞水母和沙蜇毒素对鸡血红细胞均具有明显的溶血活性，其半溶血率HU50分别为10和10.5μg/mL；理化因素对溶血活性的影响中，温度的影响最敏感，在37℃时溶血活性最强，但是当温度超过45℃时，其溶血活性明显降低。采用离子交换层析(DEAE Sepharose Fast Flow)和凝胶过滤层析(Superdex75/200)等分离纯化方法，分别从霞水母和沙蜇毒素中分离到具有溶血活性蛋白CnPH和SmTX,其对鸡血红细胞的半溶血率HU50分别为5和70μg/mL。
3.研究了水母毒素的体外抗氧化活性，并对沙蜇毒素的抗氧化活性蛋白进行分离纯化。结果表明，霞水母和沙蜇毒素对羟自由基(·OH）的半清除率EC50分别为200和500μg/mL，对超氧阴离子(O2-·)的半清除率EC50分别约为5.5和75μg/mL。利用硫酸铵分级沉淀和凝胶过滤(Superdex75)两种分离方法，从沙蜇毒素中分离到分子量为90kDa具有抗氧化活性的蛋白SmP90，该蛋白具有明显的清除超氧阴离子自由基(O2-·)的能力，其半清除率EC50约为16μg/mL，并且测定其N末端序列为：Asn-Leu-Asp-Thr-Pro-Tyr-Cys-Phe-Tyr-Ser-Gly-Asp-Tyr-Gly-Gly。
4.以沙蜇毒素蛋白为载体，利用Shotgun蛋白质组学方法首次对水母毒素的蛋白组成进行分析，共鉴定出181种蛋白。利用生物信息学方法将鉴定出来的蛋白质进行信息学分析，包括按照生物学进程、细胞组分及分子功能将其分别分为13、9和7类；并且从生物学通路里映射到33条相应的通路，在此基础上构建了基因网络来分析这些蛋白质可能存在的酶-酶、蛋白-蛋白以及基因表达之间的相互关系。
通过本文的研究表明：霞水母和沙蜇毒素具有明显溶血活性和抗氧化活性，经过分离纯化，分别从霞水母和沙蜇毒素内分离到溶血活性蛋白CnPH和SmTX及抗氧化活性蛋白SmP90；并应用蛋白质组学方法分析了沙蜇刺丝囊细胞毒素蛋白，共鉴定出181种蛋白质并且利用生物信息学方法对其进行了统计分析。
Jellyfish is widely distributed in almost every ocean from the surface to the deepsea with plenty of species, which is a very rich kind of marine biological resource.Jellyfish venom, primarily in the nematocyst of the tentacles, is a complex mixturewith novel structure and many biological activities. However, the structure andactivities vary a lot among species. In present study, jellyfish Cyanea sp. andStomolophus meleagris, which have often bloomed in the coast of china in recentyears, were chosen as the research object to study the extraction, preparing,bioactivities, isolation and proteomic analysis of the jellyfish venom and the resultsare as follows:
1. An extraction method of the jellyfish venom was studied by usingMini-Beadbeater to disrupt the nematocysts and extract the venom. Firstly, weprepared the nematocysts from the tentacles and then studied the protection of thevenom proteins by the protein inhibitors in the extraction. The results showed that1mmol/L-1EDTA and4μg/mL Pepstantin A could protect the biological activityvery well.
2. Hemolytic activity and the influencing factors on the hemolytic activity of thejellyfish venom were studied, including temperature, pH, and metal ion and so on.In addition, we have also purified two hemolytic proteins from the venom. Theresults showed that both jellyfish venoms from Cyanea sp. and Stomolophusmeleagris were hemolytic to the chicken red blood cells with the HU50of10and10.5μg/mL. Temperature was shown to be one of the most sensitive factors to thehemolytic activity of the venom. The optimal temperature for the hemolyticactivity of the venom was37°C, however, it decreased seriously when the temperature increased higher than45°C. An anion-exchange chromatographycolumn packed with DEAE Sepharose Fast Flow and a size-exclusionchromatography column with Superdex75/200were used to purify the hemolyticproteins. Finally, we have isolated two hemolytic proteins, CnPH and SmTX, withthe HU50of5and70μg/mL, approximately.
3. The in vitro antioxidant activities of jellyfish venom were investigated and theresults showed that both venom proteins from Cyanea sp. and Stomolophusmeleagris had antioxidant activity including hydroxyl radical scavenging activityand superoxide anion radical scavenging activity. The EC50of the hydroxyl radicalscavenging activity and superoxide anion radical scavenging activity of the venomproteins from Cyanea sp. and Stomolophus meleagris were200and500,5.5and75μg/mL. A90kDa antioxidant protein, which was named as SmP90, wassuccessfully isolated from the nematocyst venom proteins of jellyfishStomolophus meleagris by50%ammonium sulphate precipitation and gelfiltration chromatogram, superdex75. SmP90had superoxide anion radicalscavenging activity with the EC50of16μg/mL and the N-terminal amino acidssequences of Asn-Leu-Asp-Thr-Pro-Tyr-Cys-Phe-Tyr-Ser-Gly-Asp-Tyr-Gly-Gly.
4. Shotgun proteomic analysis of the nematocyst proteins was carried out by nanoliquid chromatography tandem mass spectrometry (nanoLC-MS/MS) for the firsttime. A total of181proteins had been identified. Bioinformatic analysis was alsoapplied to better understand the identified proteins. In the Gene Ontology (GO)annotation, all the identified proteins were classified into13,9and7groupsaccording to biological process, cellular component and molecular function,respectively. Pathways analysis of the identified proteins was conducted with33corresponding pathways found. On the basis of pathway analysis, we constructedthe gene network to analyze the relationship of those genes each other, whichcontained enzyme-enzyme relation, protein-protein interaction and geneexpression interaction.In present study, the nematocyst venom from the jellyfish Cyanea sp. andStomolophus meleagris had hemolytic activity and antioxidant activity. Two hemolytic proteins, CnPH and SmTX, and an antioxidant protein, SmP90, wereisolated from the jellyfish venom proteins. Shotgun proteomic analysis of thenematocyst proteins was carried out to gain comprehensive insight into the proteincomponent and search some novel bioactive molecules. A total of181proteins hadbeen identified and the bioinformatic analysis those identified proteins were carriedout, which would be significant to the deeper study and application of the jellyfishvenom in the future.
1.对水母毒素的提取制备方法进行了研究。以霞水母为研究对象，首先制备水母的刺丝囊细胞，然后采用组织研磨器Mini-beadbeater破碎刺丝囊细胞提取毒素，并且研究了提取过程中蛋白酶抑制剂对霞水母毒素溶血活性的影响，结果发现1mmol/LEDTA和4μg/mL PepstantinA能够有效地保护霞水母毒素的溶血活性。
2.研究了水母毒素的溶血活性，并经纯化得到具有溶血活性的蛋白。霞水母和沙蜇毒素对鸡血红细胞均具有明显的溶血活性，其半溶血率HU50分别为10和10.5μg/mL；理化因素对溶血活性的影响中，温度的影响最敏感，在37℃时溶血活性最强，但是当温度超过45℃时，其溶血活性明显降低。采用离子交换层析(DEAE Sepharose Fast Flow)和凝胶过滤层析(Superdex75/200)等分离纯化方法，分别从霞水母和沙蜇毒素中分离到具有溶血活性蛋白CnPH和SmTX,其对鸡血红细胞的半溶血率HU50分别为5和70μg/mL。
3.研究了水母毒素的体外抗氧化活性，并对沙蜇毒素的抗氧化活性蛋白进行分离纯化。结果表明，霞水母和沙蜇毒素对羟自由基(·OH）的半清除率EC50分别为200和500μg/mL，对超氧阴离子(O2-·)的半清除率EC50分别约为5.5和75μg/mL。利用硫酸铵分级沉淀和凝胶过滤(Superdex75)两种分离方法，从沙蜇毒素中分离到分子量为90kDa具有抗氧化活性的蛋白SmP90，该蛋白具有明显的清除超氧阴离子自由基(O2-·)的能力，其半清除率EC50约为16μg/mL，并且测定其N末端序列为：Asn-Leu-Asp-Thr-Pro-Tyr-Cys-Phe-Tyr-Ser-Gly-Asp-Tyr-Gly-Gly。
4.以沙蜇毒素蛋白为载体，利用Shotgun蛋白质组学方法首次对水母毒素的蛋白组成进行分析，共鉴定出181种蛋白。利用生物信息学方法将鉴定出来的蛋白质进行信息学分析，包括按照生物学进程、细胞组分及分子功能将其分别分为13、9和7类；并且从生物学通路里映射到33条相应的通路，在此基础上构建了基因网络来分析这些蛋白质可能存在的酶-酶、蛋白-蛋白以及基因表达之间的相互关系。
通过本文的研究表明：霞水母和沙蜇毒素具有明显溶血活性和抗氧化活性，经过分离纯化，分别从霞水母和沙蜇毒素内分离到溶血活性蛋白CnPH和SmTX及抗氧化活性蛋白SmP90；并应用蛋白质组学方法分析了沙蜇刺丝囊细胞毒素蛋白，共鉴定出181种蛋白质并且利用生物信息学方法对其进行了统计分析。
Jellyfish is widely distributed in almost every ocean from the surface to the deepsea with plenty of species, which is a very rich kind of marine biological resource.Jellyfish venom, primarily in the nematocyst of the tentacles, is a complex mixturewith novel structure and many biological activities. However, the structure andactivities vary a lot among species. In present study, jellyfish Cyanea sp. andStomolophus meleagris, which have often bloomed in the coast of china in recentyears, were chosen as the research object to study the extraction, preparing,bioactivities, isolation and proteomic analysis of the jellyfish venom and the resultsare as follows:
1. An extraction method of the jellyfish venom was studied by usingMini-Beadbeater to disrupt the nematocysts and extract the venom. Firstly, weprepared the nematocysts from the tentacles and then studied the protection of thevenom proteins by the protein inhibitors in the extraction. The results showed that1mmol/L-1EDTA and4μg/mL Pepstantin A could protect the biological activityvery well.
2. Hemolytic activity and the influencing factors on the hemolytic activity of thejellyfish venom were studied, including temperature, pH, and metal ion and so on.In addition, we have also purified two hemolytic proteins from the venom. Theresults showed that both jellyfish venoms from Cyanea sp. and Stomolophusmeleagris were hemolytic to the chicken red blood cells with the HU50of10and10.5μg/mL. Temperature was shown to be one of the most sensitive factors to thehemolytic activity of the venom. The optimal temperature for the hemolyticactivity of the venom was37°C, however, it decreased seriously when the temperature increased higher than45°C. An anion-exchange chromatographycolumn packed with DEAE Sepharose Fast Flow and a size-exclusionchromatography column with Superdex75/200were used to purify the hemolyticproteins. Finally, we have isolated two hemolytic proteins, CnPH and SmTX, withthe HU50of5and70μg/mL, approximately.
3. The in vitro antioxidant activities of jellyfish venom were investigated and theresults showed that both venom proteins from Cyanea sp. and Stomolophusmeleagris had antioxidant activity including hydroxyl radical scavenging activityand superoxide anion radical scavenging activity. The EC50of the hydroxyl radicalscavenging activity and superoxide anion radical scavenging activity of the venomproteins from Cyanea sp. and Stomolophus meleagris were200and500,5.5and75μg/mL. A90kDa antioxidant protein, which was named as SmP90, wassuccessfully isolated from the nematocyst venom proteins of jellyfishStomolophus meleagris by50%ammonium sulphate precipitation and gelfiltration chromatogram, superdex75. SmP90had superoxide anion radicalscavenging activity with the EC50of16μg/mL and the N-terminal amino acidssequences of Asn-Leu-Asp-Thr-Pro-Tyr-Cys-Phe-Tyr-Ser-Gly-Asp-Tyr-Gly-Gly.
4. Shotgun proteomic analysis of the nematocyst proteins was carried out by nanoliquid chromatography tandem mass spectrometry (nanoLC-MS/MS) for the firsttime. A total of181proteins had been identified. Bioinformatic analysis was alsoapplied to better understand the identified proteins. In the Gene Ontology (GO)annotation, all the identified proteins were classified into13,9and7groupsaccording to biological process, cellular component and molecular function,respectively. Pathways analysis of the identified proteins was conducted with33corresponding pathways found. On the basis of pathway analysis, we constructedthe gene network to analyze the relationship of those genes each other, whichcontained enzyme-enzyme relation, protein-protein interaction and geneexpression interaction.In present study, the nematocyst venom from the jellyfish Cyanea sp. andStomolophus meleagris had hemolytic activity and antioxidant activity. Two hemolytic proteins, CnPH and SmTX, and an antioxidant protein, SmP90, wereisolated from the jellyfish venom proteins. Shotgun proteomic analysis of thenematocyst proteins was carried out to gain comprehensive insight into the proteincomponent and search some novel bioactive molecules. A total of181proteins hadbeen identified and the bioinformatic analysis those identified proteins were carriedout, which would be significant to the deeper study and application of the jellyfishvenom in the future.
引文
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