水母刺丝囊毒素的提取、溶血活性和毒性的研究
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摘要
水母毒素是一类结构独特而新颖的蛋白质,具有多种生物活性,是潜在的海洋药物先导化合物。本文系统研究了水母毒性细胞——刺丝囊的性质、刺丝囊毒素的提取及刺丝囊毒素的溶血活性和毒性等方面的内容。主要结果如下:
通过显微镜、扫描电镜及透射电镜等技术分别对霞水母(Cyanea Sp)、海蜇(Rhopilema esculentum Kishinouye),沙蜇(Stomolophus nomurai Kishinouye),三种水母触手中所含刺丝囊的形态特点进行了分析,发现三种水母触手中含有不同形状和大小的刺丝囊。这一实验结果可以为三种水母的鉴别提供帮助。在提取毒素过程中,应用珠磨式组织研磨器破碎刺丝囊,快速简便,可有效提取刺丝囊毒素。
霞水母刺丝囊毒素具有很强的溶血活性。4-37℃范围内毒素的溶血活性与温度变化密切相关:在4℃下,毒素不能发生溶血,在37℃反应45min,毒素的溶血活性达到最大值。毒素溶血活性对pH敏感,最佳pH为7.8。胰蛋白酶能明显降低毒素的溶血活性。毒素的溶血活性具有二价金属离子依赖性,Ca~(2+)可能是毒素溶血活性的重要活化剂,但是Ca~(2+)对毒素溶血活性没有稳定作用。适量的添加EDTA,NaCl及GSH对毒素的溶血活性具有稳定作用,正交实验表明,毒素中含有1.0mM GSH,5.0mM NaCl时对溶血活性的稳定作用达到最佳效果。鞘磷脂可能是溶血分子的特异性作用靶点。
水母刺丝囊毒素与触手中提取的毒素含有明显不同的蛋白组成,三种水母毒素的蛋白组成也存在明显的差别。刺丝囊毒素的溶血活性强于触手中提取的毒素。溶血活性强弱顺序:海蜇>沙蜇>霞水母。
霞水母刺丝囊毒素的毒性研究表明,毒素表现出明显的神经毒性作用。毒素对草鱼的毒性具有剂量依赖性。霞水母刺丝囊毒素的致死毒性对热的稳定性要比已报道的其它水母毒素强,65℃加热20min毒素仍检测到明显的致死毒性,80℃加热40min或100℃加热20min可使毒素完全失活。毒性对pH敏感,最佳pH为7.8。胰蛋白酶对毒素的毒性具有明显的抑制作用。毒素的毒性在-80℃稳定。通过DEAE-Sepharose Fast Flow和Sephadex G-100分离霞水母刺丝囊毒素,主要得到两条蛋白谱带,分子量分别为60kDa和47kDa。毒素中的溶血活性成分不具有致死毒性,其等电点大于7.8。而毒素中的毒性成分等电点低于7.8,而溶血活性成分的等电点高于7.8。
通过本文的研究表明,不同水母所含刺丝囊种类不同,提取的水母刺丝囊毒素具有明显的溶血活性和毒性。本研究为进一步对水母毒素进行分离纯化,毒理药理学研究打下了基础。
Jellyfish toxins are one important protein group of marine medicine leads with unique and novel structures.The characterization of the nematocysts-the jellyfish toxic cells,the extraction of the nematocyst venom and the hemolytic and toxic activity of the nematocyst venom were investigated in this study.Main results are as follows:
1.The microscopic,scan and ultrastructure viewes of the nematocysts of Cyanea Sp, Rhopilema esculentum Kishinouye and Stomolophus nomurai Kishinouye exhibited that these jellyfishes contained different types of nematocysts.The mini-bead beater could be used to rupture the nematocysts of jellyfish and to extract the nematocyst venom.
2.The Cyanea sp nematocyst venom had strong hemolytic activity.The hemolytic activity was temperature-dependent:at 4℃,no hemolysis occurred;incubation at 37℃for 45min,the hemolytic activity reached to the highest.The venom was sensitive to pH value.The strongest hemolytic activity was found at pH 7.8. Trypsin could greatly inhibit the hemolytic activity.The hemolytic activity was divalent-cations dependent.Ca~(2+) might be an important activator for the hemolysis of the venom,however,it had no effect on the stability of the venom.The presence of EDTA,NaCl and GSH was benefit for the stability of the hemolytic activity.According to the results of orthogonal test,GSH had the strongest effect on the hemolytic activity.
3.The protein composition had significant difference between the tentacle extract and the nematocyst extract.The nematocyst extract exhibited sronger hemolytic activity than the tentacle extract.The hemolytic activity:Rhopilema esculentum Kishinouye> Cyanea Sp> Stomolophus nomurai Kishinouye.
4.The Cyanea sp nematocyst venom was toxic to fish,producing typical neurotoxin toxicity.The ID_(50) was about 0.6μg protein/g·fish.Toxic venom was stable when kept at -80℃,but a great loss of the lethality was observed when the venom was stored at -20℃.The toxic activity of the nematocyst venom of Cyanea sp was stabler than other reported jellyfish toxins.Heating at 60℃for 20 min,the venom still had significant lethal effect.Heating at 80℃for 40min or 100℃for 20min totally inactivated the toxicity of the venom.The venom was hydrolyzed by a proteolytic enzyme,trypsin.
5.The study on the separation of the Cyanea sp nematocyst venom exhibited that the hemolytic molecular was not toxic to fish.The isoelectric point of the hemolytic component was higher than 7.8 while the toxic component' was lower than 7.8. The separation of the Cyanea sp nematocyst venom using DEAE-Sepharose Fast Flow and Sephadex G-100 mainly yielded two protein bands with molecular weights of 60 kDa and 47 kDa.
In conclusion,the different species of jellyfishes contained different toxic nematocysts.The Cyanea sp nematocyst venom had strong hemolytic and toxic activity.The present study established foundations for the separation and characterization of the jellyfish venom,the study on the mechanisms of the venom action and the pharmacology,and the theatment on the jellyfish stings in the future.
通过显微镜、扫描电镜及透射电镜等技术分别对霞水母(Cyanea Sp)、海蜇(Rhopilema esculentum Kishinouye),沙蜇(Stomolophus nomurai Kishinouye),三种水母触手中所含刺丝囊的形态特点进行了分析,发现三种水母触手中含有不同形状和大小的刺丝囊。这一实验结果可以为三种水母的鉴别提供帮助。在提取毒素过程中,应用珠磨式组织研磨器破碎刺丝囊,快速简便,可有效提取刺丝囊毒素。
霞水母刺丝囊毒素具有很强的溶血活性。4-37℃范围内毒素的溶血活性与温度变化密切相关:在4℃下,毒素不能发生溶血,在37℃反应45min,毒素的溶血活性达到最大值。毒素溶血活性对pH敏感,最佳pH为7.8。胰蛋白酶能明显降低毒素的溶血活性。毒素的溶血活性具有二价金属离子依赖性,Ca~(2+)可能是毒素溶血活性的重要活化剂,但是Ca~(2+)对毒素溶血活性没有稳定作用。适量的添加EDTA,NaCl及GSH对毒素的溶血活性具有稳定作用,正交实验表明,毒素中含有1.0mM GSH,5.0mM NaCl时对溶血活性的稳定作用达到最佳效果。鞘磷脂可能是溶血分子的特异性作用靶点。
水母刺丝囊毒素与触手中提取的毒素含有明显不同的蛋白组成,三种水母毒素的蛋白组成也存在明显的差别。刺丝囊毒素的溶血活性强于触手中提取的毒素。溶血活性强弱顺序:海蜇>沙蜇>霞水母。
霞水母刺丝囊毒素的毒性研究表明,毒素表现出明显的神经毒性作用。毒素对草鱼的毒性具有剂量依赖性。霞水母刺丝囊毒素的致死毒性对热的稳定性要比已报道的其它水母毒素强,65℃加热20min毒素仍检测到明显的致死毒性,80℃加热40min或100℃加热20min可使毒素完全失活。毒性对pH敏感,最佳pH为7.8。胰蛋白酶对毒素的毒性具有明显的抑制作用。毒素的毒性在-80℃稳定。通过DEAE-Sepharose Fast Flow和Sephadex G-100分离霞水母刺丝囊毒素,主要得到两条蛋白谱带,分子量分别为60kDa和47kDa。毒素中的溶血活性成分不具有致死毒性,其等电点大于7.8。而毒素中的毒性成分等电点低于7.8,而溶血活性成分的等电点高于7.8。
通过本文的研究表明,不同水母所含刺丝囊种类不同,提取的水母刺丝囊毒素具有明显的溶血活性和毒性。本研究为进一步对水母毒素进行分离纯化,毒理药理学研究打下了基础。
Jellyfish toxins are one important protein group of marine medicine leads with unique and novel structures.The characterization of the nematocysts-the jellyfish toxic cells,the extraction of the nematocyst venom and the hemolytic and toxic activity of the nematocyst venom were investigated in this study.Main results are as follows:
1.The microscopic,scan and ultrastructure viewes of the nematocysts of Cyanea Sp, Rhopilema esculentum Kishinouye and Stomolophus nomurai Kishinouye exhibited that these jellyfishes contained different types of nematocysts.The mini-bead beater could be used to rupture the nematocysts of jellyfish and to extract the nematocyst venom.
2.The Cyanea sp nematocyst venom had strong hemolytic activity.The hemolytic activity was temperature-dependent:at 4℃,no hemolysis occurred;incubation at 37℃for 45min,the hemolytic activity reached to the highest.The venom was sensitive to pH value.The strongest hemolytic activity was found at pH 7.8. Trypsin could greatly inhibit the hemolytic activity.The hemolytic activity was divalent-cations dependent.Ca~(2+) might be an important activator for the hemolysis of the venom,however,it had no effect on the stability of the venom.The presence of EDTA,NaCl and GSH was benefit for the stability of the hemolytic activity.According to the results of orthogonal test,GSH had the strongest effect on the hemolytic activity.
3.The protein composition had significant difference between the tentacle extract and the nematocyst extract.The nematocyst extract exhibited sronger hemolytic activity than the tentacle extract.The hemolytic activity:Rhopilema esculentum Kishinouye> Cyanea Sp> Stomolophus nomurai Kishinouye.
4.The Cyanea sp nematocyst venom was toxic to fish,producing typical neurotoxin toxicity.The ID_(50) was about 0.6μg protein/g·fish.Toxic venom was stable when kept at -80℃,but a great loss of the lethality was observed when the venom was stored at -20℃.The toxic activity of the nematocyst venom of Cyanea sp was stabler than other reported jellyfish toxins.Heating at 60℃for 20 min,the venom still had significant lethal effect.Heating at 80℃for 40min or 100℃for 20min totally inactivated the toxicity of the venom.The venom was hydrolyzed by a proteolytic enzyme,trypsin.
5.The study on the separation of the Cyanea sp nematocyst venom exhibited that the hemolytic molecular was not toxic to fish.The isoelectric point of the hemolytic component was higher than 7.8 while the toxic component' was lower than 7.8. The separation of the Cyanea sp nematocyst venom using DEAE-Sepharose Fast Flow and Sephadex G-100 mainly yielded two protein bands with molecular weights of 60 kDa and 47 kDa.
In conclusion,the different species of jellyfishes contained different toxic nematocysts.The Cyanea sp nematocyst venom had strong hemolytic and toxic activity.The present study established foundations for the separation and characterization of the jellyfish venom,the study on the mechanisms of the venom action and the pharmacology,and the theatment on the jellyfish stings in the future.
引文
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