【作者】 李彦；

【导师】 王士长；

【作者基本信息】 广西大学 ， 动物营养， 2004， 硕士

【摘要】 以广西北海涠洲岛特有的马尾藻为原料，探索了马尾藻多糖的提取分离及纯化方法，测定了多糖的分子量及其单糖构成。并利用马尾藻多糖作为佐剂探索其对疫苗免疫效果的影响。 1．马尾藻多糖的提取分离及纯化 采用超声波细胞破壁的方法对马尾藻进行预处理，作用时间20分钟，80℃水浴1小时，过滤，离心，浓缩，用工业酒精沉淀出多糖，干燥后得粗多糖，提取率为13.12％，比传统热提法提高8.73％，比胶体磨法提高1.39％，而提取时间分别比胶体磨法和传统热提法缩短300分钟和420分钟。粗多糖再经过滤、酶解与透析后得到多糖SP。 通过DEAE—纤维素层析对马尾藻多糖SP进行分离，得出粗多糖由SP-Ⅰ、SP-Ⅱ、SP-Ⅲ三种组分组成，其所占比例分别为31.4％、23.8％和8.1％。再经SephadexG-100凝胶柱层析进一步分离纯化得到均一的多糖组分SSP-Ⅰ、SSP-Ⅱ、SSP-Ⅲ。凝胶色谱法测定这三种多糖组分的分子量为53862、46536和49866。高效液相色谱分析SSP-Ⅰ、SSP-Ⅱ均由阿拉伯糖、木糖、葡萄糖和半乳糖，其摩尔比分别为0.012：0.18：0.676：0.144和0.015：0.178：0.685：0.122；SSP-Ⅲ由鼠李广西大学硕一!:学位论文糖、木糖、葡萄糖和半乳糖组成，摩尔比为0.039:0.1 21:0.726:0.1 14。2.马尾藻多糖佐剂对猪pRRS疫苗免疫效果的影响 用多糖sP按低剂量组(50mg)、中剂量组(1 00mg)、高剂量组(200mg)分别与猪PRRS灭活抗原配制成完全佐剂疫苗，免疫5组非猪PRRsV免疫猪，每组5头，设空白组和普通佐剂疫苗组作对照，于首免的第0天、20天、40天、50天采血，测定体液免疫和细胞免疫水平。试验结果表明:(1)各多糖佐剂组T淋巴细胞转化率(61 .4%、65%、63.4%)均高于空白组(52.8%)及普通佐剂疫苗组(59.6%)，各多糖佐剂组与空白对照组间差异显著(p<0.05);(2)各多糖佐剂组的丁淋巴细胞亚群数量均高于空白组和普通佐剂疫苗组，其中以co3的数量差异最明显(p<0 .05)，但多糖组与普通佐剂疫苗组的CDS和NK细胞亚群的测定值差异不显著。(3)间接ELASA法测定各多糖佐剂组血清中抗体的活性(0.631、1 .015、0.688)均高于两对照组(0.518、0.568)，其中1 oomg多糖佐剂组与对照组间的差异显著 (P(0 .05)。 研究结果表明:以马尾藻多糖作为佐剂即可以提高体液免疫水平，又能增强细胞免疫功能，其中100mg多糖剂量的总体效果最佳。更多还原

【Abstract】 This paper has explored the method of extraction and separation and purition of polysaccharides from Sargassum , the specific Sargassum species in WeiZhouDao, BeiHai, GuangXi, and determinated its molecular weight and components. The polysaccharides was examined for the effect of immune response as adjuvant.Part 1. Studies on the extraction and separation and purification of polysaccharides from Sargassum.It took twenty minutes to pretreat the Sargassum with ultrasonic , in order to break the cell wall, then extracting 3h at 80℃, filtrating, certrifuging .condensing, depositing the sulphated polysaccharides with alohol, and drying . The extraction rate of polysaccharides was 13. 12%, and increased respectively by 8.73% and 1.39% comparing to traditional method and Colloid milling, and decreased by 300 minuts and 420 minuts for extraction time. Then the polysaccharides SP was made by filtrating and enzymolysis.By grading the Sargassum polysaccharides SP over DEAE-cellulosecolume and through separation , we found that the polysaccharides included three components (SP-I, SP-II, SP-III), the percentage were 31.4%, 23.8% and 8.1% respectively. Then further separation and purification steps that were done through chromato graphyon SephadexG-100 column were used to obtain a fraction named SSP-I, SSP-II, SSP-III. Them molecular weight were 53862,46536 and 49866 respectively by the same way .Theirchimical ingredients and rations shown as follows, SSP-I: Ara: Xyl: Glu: Gal =0.012: 0.18: 0.676: 0. 144;SSP-II: Ara: Xyl: Glu: Gal -0.015: 0. 178:0.685 :0. 122; SSP-III: Rha: Xyl : Glu:Ara: Gal =0.039: 0. 121:0.726:0. 114. Part 2. The effect for immune response to PRRS virus vaccine asadjuvantExperimental groups were vaccimated with normal adjuvant vaccine and three kinds of complete adjuvant vaccines which used 50mg, lOOmg, 200mg polysaccharides as adjuvant respectively +inactivity PRRS antigen; every group have five non-immune pigs. Blooding and detemining its cell-immunity and humoral-immunity standard in the ON 20> 40 ., 50 day after primary vaccinate.The results showed (1)T lymphocyte transformation rate of polysaccharides groups are :61.4%, 65%, 63.4% all higher than control group 52.8% and normal adjuvant group 59.6%. And there was a obviouse defference betwen polysaccharides groups and control group(P<0. 05);(2) Qutities of T lymphocyte subsets were all higher than the other groups,the increasion of CD3 was the most obvious. But comparing of CD8 and NK cell was not significant;(3)Antibody obsordance in serum from polysaccharides groups were 0.631,1.015,0.688 .which was higher than control group 0. 518 and normal group 0. 568 determinated by the method of indirectly ELISA, and the defference was significant (P<0. 05).The studies of result demonstrated that Sargassumpolysaccharides as adjuvant could improve humoral- immunity and enhance cell-immunity. Among the three polysaccharides groups, the total result of lOOmg group was the best.更多还原