【作者】 任国艳；

【导师】 李八方；

【作者基本信息】 中国海洋大学 ， 食品科学， 2008， 博士

【摘要】 海蜇(Rhopilema esculehtum Kishinouye)是一种大型的食用水母,在我国有着广泛的资源分布,具有很高的经济价值、营养价值和药用价值。近代研究表明海蜇有治疗高血压、慢性气管炎、哮喘、胃溃疡和单纯性甲状腺肿大等作用。从海蜇的营养保健及药学功能来看,海蜇具有进一步开发和利用的价值。目前,海蜇的一些化学成分及生物活性已经被研究探讨,如蛋白,毒素,多肽和糖胺聚糖等,但关于海蜇口腕部糖蛋白的研究未见报道。本论文以青岛附近黄海海域的新鲜海蜇为原料,采用现代的分离纯化技术,仪器分析技术,药理研究技术和分子生物学技术,从海蜇口腕部分离纯化出一种新的糖蛋白(JGP-Ⅲ),并对其理化性质,清除自由基活性,免疫活性进行研究,旨在为海蜇资源的进一步开发提供理论依据和技术支持。实验主要获得如下研究结果:1利用单因素实验和响应面分析方法中的Box-Behnken试验设计,对海蜇口腕部糖蛋白的提取工艺进行优化,结果显示,糖蛋白提取的最佳工艺条件为:提取溶剂为pH值为7.26的PBS,料液比为1:4,提取时间为7h。在此基础上。进行超声辅助提取,提取的最佳工艺条件是:超声处理时间为15min,超声功率为300W,提取时间60min。在此优化条件下,目标糖蛋白的得率为9.14%。2海蜇口腕部提取液经乙醇分级沉淀,Sp Sephadex C-25阳离子交换柱层析初步分离,得到3组份,分别为JGP-Ⅱ, JGP-Ⅲ, JGP-Ⅳ,选择糖和蛋白含量及活性较高的组分JGP-Ⅲ进行进一步研究。JGP-Ⅲ经Sephacryl S300HR层析柱, Sepharose CL-6B柱层析和HPLC鉴定JGP-Ⅲ呈现单一对称的峰;经SDS-PAGE电泳后,通过PAS和考马斯亮兰两种染色方法,在凝胶上相应的位置出现了一条带。以上结果显示JGP-Ⅲ是组成均一的糖蛋白,而不是糖和蛋白的混合物。得率为0.5%(占海蜇口腕部干基重)。3 JGP-Ⅲ冻干粉是白色絮状粉末,易溶于水,不溶于乙醇、甲醇、丙酮等有机溶剂。经化学分析方法测定,JGP-Ⅲ中含有12.61%总糖,74.34%总蛋白,8.47%氨基糖,0.84%糖醛酸,1.06%硫酸根、0.92%唾液酸。JGP-Ⅲ经SDS-PAGE测定分子量为109.7kDa,经HPLC测定,其分子量为85.3 kDa,两种方法测定得到的分子量有一定差别。JGP-Ⅲ的Td值为31.06℃,Ts值为61.74℃。JGP-Ⅲ的单糖组成主要有氨基葡萄糖,氨基半乳糖,葡萄糖,甘露糖,岩藻糖和鼠李糖。氨基酸组成中甘氨酸含量最高,其次为缬氨酸、谷氨酸、丙氨酸、脯氨酸、天门冬氨酸等,缺乏色氨酸和组氨酸。JGP-Ⅲ经消除反应前后紫外吸收和氨基酸组成的变化,说明JGP-Ⅲ中有O-连接糖苷键的存在。经糖肽酶F作用后,分子量发生明显变化,说明JGP-Ⅲ中有N-连接糖苷键的存在。JGP-Ⅲ的红外图谱具有典型的糖蛋白特征,再次证明JGP-Ⅲ是一种糖和蛋白的复合物。NMR图谱显示了JGP-Ⅲ结构特点。以上结果显示JGP-Ⅲ是一种新的糖蛋白,这为JGP-Ⅲ的进一步研究提供理论依据。4试验通过化学发光的方法,检测JGP-Ⅲ具有较强的清除羟自由基和超氧自由基能力。JGP-Ⅲ的由糖部分和蛋白部分共同参与清除自由基活动;在清除自由基活动中,N-连接的寡糖链起到主要的作用;当温度达到热收缩温度61.74℃(即蛋白纤维收缩到原来的1/3)时,清除自由基能力最强,说明JGP-Ⅲ清除自由基活性与其空间三维结构有密切关系。5采用JGP-Ⅲ溶液灌胃免疫低下模型小鼠,探讨了JGP-Ⅲ免疫调节功能。结果表明,不同剂量组的JGP-Ⅲ均能提高小鼠脾指数及胸腺指数(P<0.05);显著提高荷瘤小鼠血清溶血素含量(P<0.01)和抗体形成细胞数(P<0.05);提高迟发型变态反应;促进巨噬细胞的吞噬能力(P<0.05,P<0.01)。提示JGP-Ⅲ能全面调节机体的特异性和非特异性免疫功能,从而提高机体免疫能力。6用MTT法检测了JGP-Ⅲ对从小鼠的脾脏细胞中分离的淋巴细胞的增殖活性的影响,结果发现,JGP-Ⅲ可以促进小鼠脾脏淋巴细胞的增殖活性,并且主要是通过对T淋巴细胞群起作用的,而对B淋巴细胞几乎没有影响。JGP-Ⅲ作用的最佳浓度为50μg/ml。进一步通过RT-PCR方法研究了JGP-Ⅲ对T淋巴细胞分泌的细胞因子IL-2, IL-4, IL-6和IFN-γ的影响,结果显示:细胞内的IL-2和IFN-γ的mRNA水平很快升高,而对IL-4和IL-6的作用则相对较慢,说明JGP-Ⅲ对T淋巴细胞的作用主要是作用于Thl亚细胞群。从细胞水平上揭示了JGP-Ⅲ免疫调节作用的途径。更多还原

【Abstract】 Rhopilema esculentum jellyfish, a species of large and edible jellyfish, having the high economic, nutritional and medicinal value, is a big and important fishery resource in China. It was found that jellyfish(R.e)had an effective cure for hypertension, chronic tracheitis, asthma, gastric ulcer and struma. Considering its nutrient and curative value, R. esculentum jellyfish should be further investigate and exploited for its chemical compositions and pharmacological characters. Although some bioactivity components such as protein, toxic, polypeptides and carbohydrates were researched, no report on the biological activity of glycoprotein from jellyfish (R.e) oral-arms had yet been made.Having fresh jellyfish (R. esculentum, captured in Yellow Sea near Qingdao city) as material in this dissertation and utilizing a series of modern technologies such as isolation and purification technology, instrument analysis technology, medical analysis technology and molecular biological technology, the author had systematically studied the purification, physicochemical prosperities, radical scavenging activity and immunity of glycoprotein (JGP-Ⅲ) from jellyfish(R. e) oral-arms. Main results achieved in this research as follows:1 In order to optimize extraction technology for the glycoprotein from jellyfish(R.e) oral-arms, on the basis of single factor experiments, the effects of operating conditions such as solvent pH,material ratio, ultrasonic time, ultrasonic power and extraction time on the yield of glycoprotein were analyzed by response surface methodology. The optimized extraction conditions as follows: solvent pH was 7.26, material ratio was 1:4, ultrasonic time was 15min, ultrasonic power was 300W, and extraction time was 60min. under the above mentioned conditions, the actual yield of target glycoprotein was 9.14%.2 Three grades of jellyfish glycoprotein JGP-Ⅱ, JGP-Ⅲ, JGP-Ⅳwere isolated and purified from jellyfish oral-arms through ethanol fractionated precipitation, Sp Sephadex C-25 column. The fraction (JGP-Ⅲ) with high carbohydrate content, protein content and strong radical scavenging activity was further studied. By means of Sephacryl S300HR, Sepharose CL-6B and HPLC, JGP-Ⅲwas sole peak. A blue band and a pink band appeared on the correspondence site of SDS-PAGE gel of JGP-Ⅲstained by Coomssie brilliant blue R-250 and PAS respectively. All indicated JGP-Ⅲwas not a mixture of carbohydrate and protein but a homogeneous glycoprotein. The yield of JGP-Ⅲwas 0.5%(in dry state).3 JGP-Ⅲwas white floccules, freely soluble in water, and not soluble in organic solvents such as ethanol, acetone. JGP-Ⅲcontain 12.61% total suger,74.34% protein, 8.47% amide suger,0.84% uronic acid, 1.06% sulfate group and 0.92% sialic acid. The molecular weight of JGP-Ⅲwas estimated to be 109.7kDa by SDS-PAGE and 85.3 kDa by HPLC. The denaturation temperature (Td) and shrinkage temperature (Ts) were 31.06℃and 61.74℃respectively. Monosaccharides composition of JGP-Ⅲwas Rha, Fuc, Ara, Man, Glc, GalNAc, and GlcNAc determined by GC. Amino acid composition of JGP-Ⅲwas rich in glycine, valine, glutanmic acid, alanine, proline, methionine and asparagic acid, and lacking in histidine. The existence of O-glycosidic and N-glycosidic linkage in the glycoprotein was demonstrated withβ–elimination reaction and peptide N-glycosidase F reaction. IR and NMR spectrum of the glycoprotein indicated the strcture characterization of JGP-Ⅲ. These results provided the theoretical basis for further research.4 Scavenging activities on superoxide and hydroxyl radicals of JGP-Ⅲwere estimated by chemiluminescence method, and the structure-function relationship were initial studied by chemical and enzymatic hydrolysis methods. Radical scavenging activities of JGP-Ⅲhad significant dose-dependent relationship. The carbohydrate moiety and protein moiety of JGP-Ⅲwere both involved in Scavenging radicals. The N-linked oligosaccharides played an important role in the Scavenging radicals of JGP-Ⅲ. Different structure of JGP-Ⅲhad different radicals scavenging activities. These results indicated that JGP-Ⅲhad strong free radical scavenging activities, and which had close relations with its three dimensional structure.5 The regulating effect of JGP-Ⅲon immunological function in normal and immunosuppression mice were also investigated. The results showed that JGP-Ⅲof different dosages could obviously enhance spleen and thymus indexes, increase hemolysin conten(tP<0.01)and quantity of antibody forming cells in vivo(P<0.05), heighten delayed hypersensitivity level ( P<0.05, P<0.01, and promote the phagocytosis ability of celiac macrophage(P<0.05, P<0.01). It is suggested that JGP-Ⅲcan enhance immune function by activating specific and nonspecific immunity in organism.6 The immunomodulatory of JGP-Ⅲwas investigated by the methods of molecular biology and cellular biology. JGP-Ⅲwas found to significantly increase the proliferation of total spleen lymphocytes cell populations and more strongly increases that of T cells. However, JGP-Ⅲhad less influence on the proliferation of B cells. JGP-Ⅲexerted its immunomodulating activity at an optimal dose of 50μg/mL. At this concentration, JGP-Ⅲpromoted farthest proliferation of spleen lymphocyte. Time-dependence analysis showed some differences action of JGP-Ⅲon T cells among four kinds of cytokines. IL-2 and IFN-γresponded rapidly to JGP-Ⅲ, whereas IL-4 and IL-6 were affected after a few hours treatment with JGP-Ⅲ, Accordingly, this suggested that Th1 cells, which secret IL-2 and IFN-γcytokines, were primary cellular targets directly affected by JGP-Ⅲon T lymphocyte. Whereupon, secondary response of Th2 cells related with IL-4 and IL-6 mRNA expression were followed.更多还原