【作者】 盘赛昆；

【导师】 汤坚；

【作者基本信息】 江南大学 ， 食品科学， 2009， 博士

【摘要】 鳙（Aristichthys nobilis）是我国产量较大的一种淡水鱼类食物资源,具有生长速度快、疾病少、个体大等特点。近些年来,鳙的加工利用研究取得了长足的发展,鳙产业逐步走向良性发展的道路。为了提升鳃这种加工废弃物的价值,探索鳙鳃的综合利用可行性,本文从鳙鳃中分离纯化凝集素,主要研究内容如下:从鳙鳃主要化学组成看来,鳙鳃的蛋白质含量与鱼体相当,是一种可综合利用的下脚料。分析表明,鳙鳃中含有高活性的凝集素,最优提取工艺为:缓冲体系为22.81 mmol/L的Tris-HCl缓冲液（pH 8.5）、料液比1∶6、提取时间16.43 h。在该条件下实际提取比活力为8125.49 9647.06 HU/mg。建立了一种包括DEAE-Sepharose FF离子交换层析、Sephacryl S-200 HR凝胶过滤层析及Superdex 200 10/300 GL液相快速蛋白层析技术的分离纯化工艺,从鳙鳃中分离纯化到一种对岩藻糖专一结合的凝集素,命名为GANL（gill of Aristichthys nobilis lectin）。本工艺的纯化倍数为21.4倍,蛋白质回收率为3.6%。纯化凝集素在SDS-PAGE上显示单一蛋白质条带。研究了鳙鳃凝集素的物理化学性质及其活性的影响因素,发现在供试的六种动物红细胞及人的A、B及O型血红细胞中,鳙鳃凝集素仅能凝集兔红细胞及人的O型血红细胞,表现出较强的供血动物种属专一性。鳙鳃凝集素的活性仅被岩藻糖抑制,糖结合专一性较强。鳙鳃凝集素具有较高的耐热性,最适作用温度为50℃,90℃处理30 min仍能保持相当高的活性;对酸敏感,最适作用pH为8⁹,在碱性环境中较为稳定,pH为11时还能保持一定的活性;鳙鳃凝集素的活性不依赖Ca²⁺、Mn²⁺及Mg²⁺,受还原剂β-巯基乙醇、表面活性剂SDS及胰蛋白酶水解、琥珀酰化修饰显著影响,而DMSO及三氟乙酸两种试剂对鳙鳃凝集素的活性没有显著影响。对鳙鳃凝集素的分子结构进行了初步的表征。鳙鳃凝集素是一种相对分子质量为2.20×10⁵的糖蛋白,由六个相对分子质量为3.55×10⁴的相同亚基组成。分子中糖含量为13.4 %,糖链与蛋白质部分的连接方式为O-糖苷键。鳙鳃凝集素含有0.81 %的Cys-S,结构较复杂,在非还原SDS-PAGE中显示一种梯状条带,分子中α-螺旋含量为34.8 %、β-折叠含量12.1 %、β-转角含量24.5 %和无规卷曲含量33.0 %。发现在供试的三种微藻中,鳙鳃凝集素仅能凝集雨生红球藻且这种凝集作用与藻细胞浓度有关。发现在供试的七种微生物中,鳙鳃凝集素能凝集鳗弧菌、哈维氏弧菌两种革兰氏阴性鱼类病原微生物,表现出较强的专一性。鳙鳃凝集素能抑制鳗弧菌、哈维氏弧菌的生长,这种抑制作用被0.2 mol/L的岩藻糖解除,说明鳙鳃凝集素对细菌的抑制作用与糖结合域有关。发现鳙鳃凝集素对HeLa、SKOV3、HepG2三株细胞具有一定的抑制作用,其中对HeLa细胞株的抑制作用具有剂量依赖关系,IC₅₀为11.86μg/mL,而对SKOV3及HepG2的抑制作用剂量依赖性不明显。发现鳙鳃凝集素对未经诱导的小鼠脾淋巴细胞的生长有一定的促进作用,经ConA诱导后鳙鳃凝集素的促进生长作用有明显的提高,而经LPS诱导后鳙鳃凝集素的作用并没有明显提高。因此,推测鳙鳃凝集素具有一定促进T细胞有丝分裂的作用,而对B细胞没有作用。更多还原

【Abstract】 Bighead carp （Aristichthys nobilis） is one of freshwater fish food resources with very large population. The bighead carp has a tremendous growth rate and disease resistant. Adults can be quite large, making it a lucrative aquaculture fish. In recent years, the research of utilization of bighead carp has made considerable development. Bighead carp industry is gradually moving towards healthy development. In order to explore the feasibility of the comprehensive utilization of gills from bighead carp, enhance the value of this processing waste, this article was described the purification and characterization of a lectin from gill of bighead carp. The main research contents are as follows:The chemical composition analysis of the gill and body of bighead carp showed that the protein content was nearly equal to the body. Therefore, the gill of bighead carp was a available scraps for comprehensive utilization.The optimal extraction process of the lectin from gill of bighead carp as follows: the buffer system as 22.81 mmol/L of Tris-HCl buffer （pH 8.5）, solid-liquid ratio 1:6, extraction time 16.43 h. Under these conditions, the actual extraction of specific activity was 8125.49 9647.06 HU/mg.A lectin with fucose-specific was purified from the crude extraction of gill from bighead carp. The purification procedure consisted of separation on a DEAE-Sepharose FF ion exchange column, followed by gel filtration chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Superdex 200 10/300 GL columns. The purified lectin was designated as GANL （gill of Aristichthys nobilis lectin）. The purification fold of this procedure was 21.2 times, the protein recovery was 3.6%. The purified lectin showed single protein band under SDS-PAGE.The studies of physical and chemical properties review that for the six tested animal erythrocytes and human A, B and O-type red blood cells, GANL only agglutinated rabbit native erythrocytes and human O-type red blood cells. The hemagglutination activity showed strong blood supply animal species specificity. Hemagglutination activity of GANL was not inhibited by any of the monosaccharides, disaccharide and glycoproteins tested except for fucose, showing a strong sugar-binding specificity. Our results suggest that the optimal temperature for GANL is close to 50℃. We also found that some original activity was maintained even after heating to 90℃for 30 min, suggesting that this lectin protein is extremely thermostable. GANL was unstable under acid condition but stable under alkaline conditions, the optimal pH was 8⁹. We found that some original activity was maintained following incubation at pH 11 for 1 h. The hemagglutination activity of GANL was not dependent on Ca²⁺, Mn²⁺ and Mg²⁺. The hemagglutination activity of GANL was significantly affected by reducing agentβ-mercaptoethanol, detergents SDS and trypsin hydrolysis, succinyl-modified. In contrast, The hemagglutination activity of GANL was not significantly affected by DMSO and trifluoroacetic acid.GANL was a glycoprotein with a native relative molecular weight of 2.20×10⁵, composed with six identical subunit with relative molecular weight of 3.55×10⁴. The carbohydrate content of GANL was 13.4 %. The linkage of carbohydrate and protein was O-glycosidic bond between sugar and Thr. Cys-S was detected in the lectin molecule （0.81%）. The structure of GANL was complex, showing a ladder-like protein band under non-reducing SDS-PAGE. The molecules of GANL containing theα-helix 34.8 %,β-fold 12.1 %, random coil 33.0 %,β-corner 24.5 %.The studies suggested that GANL only agglutinated Haemetococcus pluvialis among the three microalgae tested. The agglutination activity showed a cell desity dependent mannar. In the seven microorganisms tested, GANL only agglutinated two kinds of Gram-negative fish pathogenic micro-organisms, Vibrio anguillarum and Vibrio harveyi. GANL had no agglutinating effect on others bacteria and fungi. Microbial agglutination of GANL also showed stronger specificity. GANL can only inhibit the growth of V. anguillarum and V. harveyi. This growth inhibition can be converted by 0.2 mol/L of fucose suggested that the microbial growth inhibition related with CRDs.GANL exerted potent antitumor activity against the HeLa cell line、SKOV3 cell line and HepG2 cell line. The antitumor activity of GANL on HeLa cell line has a dose-dependent manner. The IC₅₀ was 11.86μg/mL,while the dose-dependent manner on SKOV3 and HepG2 was not obvious.GANL was weakly mitogenic towards the murine splenocytes in comparison with ConA, a standard plant mitogen. However, we observed significant mitogenic activity towards murine splenocytes that were previously incubated with ConA. GANL had no significant mitogenic effect towards murine splenocytes previously incubated with LPS, in comparison with GANL alone. Therefore, we speculated that GANL have mitogenic effect towards T cells, but no effect on B cells.更多还原