地稔凝集素的提取及凝血活性的研究

为研究和利用地稔的促凝血活性物质,以地稔为试材,提取地稔凝集素,研究pH、温度和糖等理化因素对凝血活性的影响。结果表明:经硫酸铵沉淀及透析提取的地稔凝集素具有红细胞凝集活性;碱性条件下,抑制作用明显,而酸性条件有较强促进作用;温度对凝集素凝血活性影响很小;半乳糖有明显促进作用。     

大黄鱼C型凝集素受体Clec4e的分子鉴定及凝集特性

C型凝集素受体（C-type lectin receptor，CTLR）是可以特异性结合糖类病原体相关分子模式（pathogen-associated molecular patterns，PAMPs）的模式识别受体（pattern recognition receptors，PPRs），在先天性免疫中发挥着重要作用。为了揭示硬骨鱼CTLR的生物学功能，本研究以从大黄鱼（Larimichthys crocea）转录组数据库中筛选出的一个CTLR基因—C型凝集素结构域家族4成员E基因（C-type lectin domain family 4 member E gene，Clec4e）为研究对象，研究其分子特征、表达分布和凝集特性。结果显示，LcClec4e cDNA全长1546 bp，开放阅读框（open reading frame，ORF）771 bp，编码254个氨基酸。LcClec4e的N端有一个跨膜区，无信号肽，C端含有一个糖识别结构域（carbohydrate recognition domain，CRD），其中含有糖结合位点EPN和WFD以及6个可形成二硫键的保守半胱氨酸。系统发育分析表明，LcClec4e与多种鲈形目鱼类Clec4e具有较近的亲缘关系。荧光定量PCR结果显示，LcClec4e在所检测的10种组织中呈组成型分布，且在肝脏中表达量最高；LcClec4e在来源于大黄鱼头肾组织的原代巨噬细胞、淋巴细胞和粒细胞中均有表达，且在巨噬细胞中表达量最高；经灭活溶藻弧菌刺激后，LcClec4e在3种免疫细胞中的表达均极显著上调。原核表达的重组LcClec4e胞外段（recombinant LcClec4e-extracellular domain，rLcClec4e-ex）具有Ca2+依赖性的凝集活性，可凝集小鼠、家兔的红细胞，以及嗜水气单胞菌、变形假单胞菌、溶藻弧菌和坎氏弧菌等4种水产常见的革兰氏阴性菌。D-葡萄糖、D-果糖、D-甘露糖、D-麦芽糖、α-乳糖和脂多糖均可抑制rLcClec4e-ex对大黄鱼重要病原菌变形假单胞菌的凝集作用，说明LcClec4e可能与变形假单胞菌表面的糖类物质结合。这些研究结果提示，LcClec4e可能作为一种PPR，通过结合病原菌表面的糖类PAMPs来识别病原，参与大黄鱼抗细菌感染的免疫防御。     

The consequence of low mannose-binding lectin plasma concentration in relation to susceptibility to Salmonella Infantis in chickens

Mannose-binding lectin (MBL) is a key protein in innate immunity. MBL binds to carbohydrates on the surface of pathogens, where it initiates complement activation via the lectin-dependent pathway or facilitates opsonophagocytosis. In vitro studies have shown that human MBL is able to bind to Salmonella, but knowledge in relation to chicken MBL and Salmonella is lacking. In order to study this relation day-old chickens from two selected lines L10H and L10L, differing in MBL serum concentration, were either orally infected with S. Infantis (S.123443) or kept as non-infected controls. The differences between healthy L10H and L10L chicken sublines were more profound than differences caused by the S. Infantis infection. The average daily body weight was higher for L10H than for L10L, regardless of infection, indicating beneficial effects of MBL selection on growth. Salmonella was detected in cloacal swabs and the number of Salmonella positive chickens during the experiment was significantly higher in L10L than L10H, indicating that MBL may affect the magnitude of Salmonella colonisation in day-old chickens. MBL expression was determined in ceca tissue by real-time RT-PCR. L10H chickens showed a significantly higher relative expression than L10L at days 1 and 41 pi, regardless of infection. Finally, flow cytometric analysis of whole blood from infected chickens showed that L10H had a significantly higher count of all assessed leucocyte subsets on day 5 pi, and also a higher count of monocytes on day 12 pi than L10L. No difference was observed between infected and non-infected L10L chicken.     

尼罗罗非鱼鼠李糖凝集素 (RBL-1)在非特异性细胞防御病原菌感染中的功能

为了探究鼠李糖凝集素（Rhamnose-binding lectin, RBL）在硬骨鱼非特异性细胞防御病原菌感染中的作用,本研究以尼罗罗非鱼为研究模型,首先通过分离头肾单核/巨噬细胞进行体外菌应激实验,发现在罗非鱼两种重要的致病菌-无乳链球菌（Streptococcus agalactiae）和嗜水气单胞菌（Aeromonas hydrophila）应激后,尼罗罗非鱼L-鼠李糖凝集素1（OnRBL-1）的表达量显著上调。然后,通过荧光定量PCR（qRT-PCR）检测发现OnRBL-1重组蛋白能够调节病原菌诱导细胞炎症因子的表达,包括显著抑制菌诱导的IL-6、IL-8和TNF-α的表达,和促进IL-10和TGF-β的表达。此外,通过流式细胞术检测证实OnRBL-1具有促进单核/巨噬细胞的吞噬作用,同时增强呼吸爆发水平和上调活性氧的释放。以上研究结果表明,OnRBL-1在罗非鱼单核/巨噬细胞非特异性细胞防御中发挥重要的调节作用。本研究为探讨RBL-1在硬骨鱼宿主防御病原菌感染中的功能提供了参考,并有助于完善和丰富硬骨鱼类RBL的功能和在抗菌免疫应答中的基础理论体系,具有重要的科学意义。     

A novel mannose-binding lectin from plasma of Labeo rohita

A new mannose-binding lectin was purified from plasma of freshwater fish, rohu (Labeo rohita) by ammonium sulphate precipitation followed by DEAE-cellulose ion-exchange chromatography. The hemagglutinating activity is strong for neuraminidase-treated rabbit erythrocytes. Mannose, glucose and their derivatives inhibit hemagglutination. The apparent molecular weight was determined to be 210 kDa in native PAGE. Analysed on SDS-PAGE under reducing and non-reducing conditions, the protein migrated as a single band of mol.wt. 40,000. The lectin is acidic in nature showing a pI of 4.5. It is a glycoprotein containing complex glycan part as indicated by carbohydrate analysis using high-pressure anion exchange chromatography with a pulsed amperometric detector. The N-terminal sequence (WLNGIGTQIPMKITT) shows no significant homology with known proteins. It appears to be a C-type lectin as Ca²⁺ is essential for carbohydrate-binding activity. Methyl -α- D-mannopyranoside was found to be the most potent inhibitor among the monosaccharides and disaccharides tested. Purified lectin was found to induce intracellular superoxide production following opsonization of E. coli by its own macrophages. This revised version was published online in August 2006 with corrections to the Cover Date.     

39个牡丹品种的形态学分类研究

【目的】探究牡丹品种分类的等级和标准,进而为其品种选育和资源研究提供参考。【方法】2013-03-05,观测了39个牡丹品种的26个质量和数量性状,首先对26个性状指标进行了R型聚类分析和主成分分析,然后根据R型聚类结果,去掉相关性大的指标,再对39个牡丹品种进行了Q型聚类分析。【结果】R型聚类结果显示,花瓣基部有无色斑与色斑颜色（r=0.766）,复叶长与复叶宽（r=0.659）的相关性较大,因此Q型聚类中不考虑色斑颜色和复叶宽2个性状。主成分分析结果表明：结实率、花瓣基部色斑情况、花期、花冠情况等12个性状的贡献率较大。Q型聚类结果显示,39个牡丹品种依据结实率、花瓣基部有无色斑和花期被分为3类：结实率高且花期较早的类群,如“赵粉”、“肉芙蓉”、“迎日红”、“粉中冠”等;结实率低且花瓣基部没有色斑的类群,如“状元红”、“绿香球”、“大胡红”、“珊瑚台”等;结实率低且花瓣基部有色斑的类群,如“红宝石”、“卷叶红”、“十八号”、“菱花湛露”等。【结论】筛选出对牡丹品种分类贡献较大的13个性状,得到可以作为其形态学分类依据的性状有结实率、花瓣基部有无色斑和花期。     

锯缘青蟹血清凝集素的特性和盐析提取研究

对锯缘青蟹(Scylla serrate)血清凝集素的凝集特性进行了研究。结果显示:锯缘青蟹血清凝集素对人、兔、鼠、鸽子、中华鳖、鲫红细胞均有凝集作用,其中对小鼠红细胞凝集活性最强,血凝活性可达29;凝集素活性最适pH 6.0~7.4;盐度对凝集活力有较大影响,NaC l浓度在0.4~0.5 mol/L时基本失活;未纯化血清凝集素对Ca2+、Mg2+未表现出依赖性;凝集素活性能被N-乙酰葡萄糖胺特异性抑制。对青蟹血清凝集素进行硫酸铵分级沉淀分离后,发现凝集素活性主要分布在25%饱和度硫酸铵沉淀区,该组分凝集比活比血清高27.44倍,SDS-PAGE结果表明主要蛋白带型为79 ku、75 ku、72 ku。     

不同菜豆品种4种抗营养因子水平差异分析

菜豆中植物凝集素、皂苷、胰蛋白酶抑制剂和植酸等抗营养因子是引起菜豆食物中毒的内源物。以56份食荚菜豆品种为材料,测定了鲜荚中4种抗营养因子含量或活性。结果表明,供试菜豆品种群体4种抗营养因子水平均存在极显著差异。植物凝集素含量(均值为1.743 mg·g~(-1))和胰蛋白酶抑制剂活性(均值为1.680 mg·g~(-1))变异系数均在100%以上,品种频率分布曲线主峰明显,但在极高和极低区域均有品种间断分布,出现了品种水平的数量等级差异;皂苷含量(均值为3.730 mg·g~(-1))和植酸含量(均值为3.102 mg·g~(-1))变异系数均低于41%,品种频率分布曲线呈近正态分布,主峰明显,双尾有低频率连续分布。相关分析表明,植物凝集素含量与胰蛋白酶抑制剂活性呈极显著正相关。聚类分析将供试56份菜豆品种划分为3个品种群,近80%品种聚于抗营养因子中等水平品种群,近12%品种居于较低水平。由于菜豆植物凝集素是食用致毒性的主要内源物,同时其和胰蛋白酶抑制剂等均与菜豆田间抗病虫性密切相关,预示着筛选出极低水平植物凝集素的菜豆品种是可行的,但同时可能会降低其田间抗病虫性。     

Influence of Phytohemagglutinin on the Agronomic Performance of Beans (Phaseolus vulgaris L.)

The abundant lectin phytohemagglutinin (10 % of total seed protein) does not contain sulfur amino acids and, being a potent antimetabolite, it is responsible for the lowering of the nutritional value of bean seeds. The aim of the present work was to improve the dry bean cultivar ‘Taylor's Horticultural’ (Asgrow), by genetically introducing the lectin null (lec/lec) character from two null genotypes: ‘Pinto UI 111’ and ‘Heidi’. Thirty-seven BC²F₃ and fourteen BC₆F₅ inbred lines were evaluated in agronomical trials. Analysis of Variance (ANOVA) showed significant differences among BC₂F₃ breedings lines for all traits under evaluation. Comparison of the LedLee genotypes versus lec/lec did not show statistically significant differences in the means for the following traits: yield, yield components and percentage of protein in the seed. Fourteen BC6F5 lines, compared together with their recurrent parent ‘Taylor's Horticultural’, showed significant differences among genotypes for 1000 seed weight, protein percentage on dry matter and ash percentage. No significant differences were observed for grain yield. The data indicate that lectin removal did not have a detrimental effect on the traits evaluated.