多糖硫酸酯化修饰及其抗凝血作用研究进展

硫酸多糖是一种单糖分子上的一个或多个羟基被硫酸根取代的多糖,近年来由于其良好的抗凝血活性及其较低的副作用被人们所关注.硫酸多糖包括天然硫酸多糖和化学法硫酸化多糖两种,研究证实两者都具有较好的抗凝血活性.对于硫酸多糖的抗凝血活性的构效关系的研究是改进其功能性质的核心,也是国内外研究的热点.本文综述了硫酸多糖的硫酸化方法,硫酸多糖的抗凝血活性以及机理,并对硫酸多糖的研究方向进行了展望.     

富含多糖草莓果实总RNA提取方法的改进

以草莓果实为模式实验材料,将Chomczynski提出的常规RNA提取方法与Kenneth等提出的改进方法相结合,并做了进一步改进,建立了一种简单实用的从富舍多糖的植物材料中提取总RNA的方法。先利用冷的丙酮去除色素类物质,再利用乙二醇丁醚去除多糖,从而有效克服了从富含色素和多糖娄物质的植物材料中提取RNA的困难;获取的RNA样品在纯度和浓度上都可以满足PCR及Northern杂交等分子生物学实验的要求。     

脑膜炎球菌多糖疫苗培养基的标准化研究

目的用干粉原料完全替代以消化液为主要原料的培养基配方,实现脑膜炎球菌多糖疫苗培养基的标准化生产。方法用筛选改良的酪蛋白酸水解物干粉替代50%盐酸酪蛋白水解液,以改良酵母浸出粉替代酵母透析液和酵母浸出粉,适当调整培养基配方,改进并稳定培养基制备工艺,确定适宜质量指标,并对各项质量指标数据进行分析。结果改进后的脑膜炎球菌多糖疫苗干粉培养基有效地降低了批间差异,各项质量指标均符合脑膜炎球菌培养要求,培养基的各项质量指标更加稳定可控,在生产中得到了良好、稳定的生长结果。结论用干粉原料完全替代脑膜炎球菌多糖疫苗培养基中的消化液和酵母透析液是成功的,同时改良的干粉培养基进一步明确了配方标准,使原料准备、制备工艺、质量指标控制更加标准化,有效提高了培养基质量,有利于规模化生产,促进了脑膜炎球菌多糖疫苗生产用培养基的标准化工作。     

适于核桃基因标记的DNA提取方法

为了对核桃(Juglans regia L．)特异性状的相关基因进行分子标记研究,采用CTAB法和改进的CTAB法,丛叶片中提取核桃基因组DNA,比较其分离效果。结果表明,常规的CTAB法不能有效去除多糖,而改进CTAB法不论老叶新叶都能有效去除细胞内多糖和多酚等杂质对模板DNA的污染,获得的DNA纯度高,可进一步用于核桃分子生物学的操作。     

改良尿素-氯化锂方法提取成熟小麦种子总RNA

为了分离提取成熟小麦种子总RNA,去除多糖等杂质的严重干扰,本实验对尿素氯化锂分离RNA的方法做了三个方面的改进:一、去除成熟种子的胚(含大量麦胚凝集素等糖蛋白),发现裂解物的黏稠度明显下降;二、在裂解粗提物中加入CTAB至终浓度为02%,可去除大部分多糖;三、异丙醇沉淀RNA之后,充分溶解,离心去除残留的不溶性多糖等杂质。结果表明:所提取的成熟小麦种子RNA的纯度和产率都有明显提高 。     

硝普钠对香菇多糖代谢及关键酶基因表达的影响

香菇多糖是香菇中特有的生物活性成分。硝普钠在调控食用菌生长方面具有重要影响,但其对香菇生长及多糖合成的影响尚不清楚。本文以香菇新808为研究材料,通过添加不同浓度硝普钠作为外源一氧化氮对新808菌丝的生物量、生长速率、香菇多糖含量、香菇多糖代谢关键酶活性及其表达调控基因相对表达量进行综合研究。结果表明：硝普钠浓度为100 μmol/L可显著提高香菇菌丝生物量,与对照组相比增加了1.61倍;生长速率与对照组无显著差异;香菇菌丝的香菇多糖含量显著提高,为对照组的3.71倍;漆酶、锰过氧化物酶和木质素过氧化物酶活性显著提高,分别为对照组的1.73倍、2.23倍和1.55倍;与多糖合成有关的辅助模块酶基因LENED_010207、碳水化合物结合模块基因LENED_012941和多糖裂解酶基因LENED_004566基因相对表达量有所上调,其中LENED_010207基因上调最明显,而多糖合成关键酶基因UDP-葡萄糖焦磷酸化酶基因(UGP)、葡萄糖磷酸变位酶基因(PGM)与葡萄糖磷酸异构酶基因(PGI)相对表达量均显著下调。在100 μmol/L浓度硝普钠处理下,香菇生物量、多糖含量、多糖代谢关键酶活性及部分多糖合成关键酶的基因相对表达量均显著高于对照组,表明添加适宜浓度的硝普纳能有效地促进香菇多糖的合成,提高香菇多糖的含量。本文为进一步探索香菇多糖的代谢通路提供了理论依据,为选育高多糖含量的香菇新品种提供了新思路,对改进香菇栽培技术有一定的参考意义。     

百合花瓣总RNA提取方法的研究

以亚洲百合品种Pollyanna和东方百合品种Sorbonne为试材,在比较异硫氰酸胍法、SDS/酚法与CTAB-L i Cl法提取总RNA效果的基础上,针对百合组织中富含多糖的特点,在Sorbonne提取RNA中加入特殊除多糖步骤,改进了CTAB- L i Cl法.结果表明,改进CTAB- L i Cl法能有效去除多糖,提取到的RNA2 8S r RNA亮度约为18S r RNA的2倍,A2 6 0 / 2 80介于1.8～2 .0之间,A2 6 0 / 2 30为2 .0 ,Pollyanna RNA产率为36 .3μL·g- 1 ,Sor-bonne RNA产率为10 .2 μL·g- 1 ,经RT- PCR获得了特异性条带,说明用改进CTAB- L i Cl法从百合花瓣中提取到的RNA质量好、产率高、完整性强,完全适合于进一步的分子生物学研究.     

C群脑膜炎球菌多糖纯化方法的改进

将C群脑膜炎球菌粗多糖的纯化改进为用1:1容量冷酚提取的生产工艺。结果表明,改进后的方法去除蛋白质效果同样能够达到《中国药典》2005版(三部)的要求,总体结果优于改进前。同时能扩大处理量而降低了生产成本,适合规模化生产。     

伤寒Vi多糖疫苗大规模生产中的工艺改进

对伤寒Ｖｉ多糖疫苗大规模生产进行了三项工艺改进：１．将液体菌种由静止期接种大罐改为对数生长期接种大罐,大罐培养时间可缩短３－４小时。２．２％（Ｖ／Ｖ）甲醛溶液杀菌时间由５℃６小时改为２０－２５℃２小时,能保证杀菌完全。３．用冷酚精制Ｖｉ多糖由常规法改为乳化法,可减少精制次数及冷酚用量,提高多糖收率     

小鼠巨噬细胞吞噬功能测定方法的改进及应用(英文)

对巨噬细胞吞噬鸡红细胞活性的体内检测方法进行改进.收集小鼠腹腔巨噬细胞,加入不同浓度甘草多糖、鸡红细胞于37℃共同孵育1 h,在倒置显微镜下观察吞噬状态,统计吞噬百分率和吞噬指数.并将改进后的体外吞噬方法用于检测酵母多糖对小鼠腹腔巨噬细胞形态和吞噬功能的影响.结果表明:改进后的体外吞噬法测定的结果与传统的体内吞噬法比较,两者具有显著的相关性(n=6,P<0.01).酵母多糖对巨噬细胞刺激1 h后,与对照组相比,其吞噬功能显著性增强;巨噬细胞形态上出现被活化的特征.应用改进后的方法研究巨噬细胞吞噬鸡红细胞,不需染色,在模拟生理条件下进行,保持了细胞活性,有简便、成本低以及准确率高的特点,适用于普通实验室的科研和教学.     

聚丙烯酰胺微生物降解研究进展

聚丙烯酰胺是一类重要的水溶性高分子聚合物,已广泛应用到工农业生产的各个领域和人们的日常生活中。由于具有良好的理化特性,一直被认为是安全、无毒和稳定的,所以有关其在自然界中的降解及其可能产生毒性的研究在很长一段时期内被忽视。事实上,聚丙烯酰胺在环境中的残留、迁移、降解对环境具有潜在危害性。目前,其应用范围和规模正呈现快速增长趋势,而其研究多集中在其合成和应用方面,对聚丙烯酰胺的降解尤其是生物降解研究极少。     

Hlad Immobilization in Polyacrylamide Gels

The immobilization of horse liver alcohol dehydrogenase in a cross-linked acrylamide-N,N¹-methylenebisacrylamide copolymer gel is described. The influence of monomer concentration, the degree of cross-linking and the polymerization technique on enzyme entrapment is studied. A bead-polymerization process produced the most useful and stable immobilized enzyme preparations.     

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.     

Hlad Immobilization in Polyacrylamide Gels

The immobilization of horse liver alcohol dehydrogenase in a cross-linked acrylamide-N,N¹-methylenebisacrylamide copolymer gel is described. The influence of monomer concentration, the degree of cross-linking and the polymerization technique on enzyme entrapment is studied. A bead-polymerization process produced the most useful and stable immobilized enzyme preparations.     

Polyacrylamide Gel Electrophoresis of Plasteins

A peptic hydrolysate of soybean protein was filtered with Sephadex G–25 and was separated approximately into four fractions (I, II, II, and IV in the order of mol. wt.). Fraction II (av. mol. wt: 1043) and III (av. mol. Wt.: 685) were more plastein-productive than others. When plastein produced from Fraction II with Nagarse was investigated by plate electrophoresis using 7.5% polyacrylamide-gel, the upper limit of the molecular weight was found to be about 25,000. A similar result was obtained also with Fraction III. The increase of molecular weight in the course of the plastein formation with the mixture (substrate) of Fractions II and III was shown that the final product lay mainly in a position between cytochrome c (mol. wt.: 11,700) and Nagarse (mol wt.: 27,600). In addition, the gel-electrophoretic experiments revealed that the most favorable condition for the plastein synthesis were pH 6.5 and 35% in substrate concentration.     

Polyacrylamide gels with modified cross-linkages

The retention of low molecular weight proteins during electrophoresis through gradient polyacrylamide gels was improved when a gradient of N,N′,N″-triallyl citric triamide (TACT) was superimposed on the gradient of acrylamide and N,N′-methylenebisacrylamide (MBA). Gels cross-linked only with N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) are soluble in dilute periodic acid or dilute aqueous solutions of bases. DHEBA cross-linked gradient gels have a smaller pore structure at high acrylamide concentrations and a more open structure at low acrylamide concentrations than gels cross-linked with MBA. Proteins labeled with tritium and carbon-14 were fractionated through DHEBA cross-linked gradient gels and the isotopes measured after solution of the gel with periodic acid. The mild solubilizing conditions enhanced isotope resolution. The characteristics of several cross-linking molecules are discussed and reasons advanced for the superiority of those with acrylamido end groups.     

Polyacrylamide gel electrophoresis on micro slabs

An electrophoretic technique using micro polyacrylamide flat gels is described and its usefulness demonstrated. The gels are vertically cast and electrophoresed in slab form (75 × 18 × 0.75 mm) in closed thin glass cells (cuvets) made from detachable microscope slides. The main features of the method are: requirement of small sample quantities (0.1–20 μg contained in <1–5 μl solution), simultaneous analysis of several samples in a single gel, relatively brief running periods, easy removal of the gel for rapid staining due to the two-piece gel mold, little pattern diffusion, convenient optical evaluation, drying, autoradiography and other contact print methods, and easy application of immunodiffusion techniques. Continuous gradient gels can be prepared. The advantage and complications of the technique are discussed and certain applications in biochemistry, clinical chemistry, and medicine are pointed out.     

阳离子聚丙烯酰胺的合成及其表征

合成了阳离子功能单体甲基丙烯酰氧乙基二甲基丁基溴化铵(DMB),并将DMB与丙烯酰胺(AM)共聚制备了阳离子聚丙烯酰胺,并用IR,NMR对其结构进行了表征。     

High Resolution Polyacrylamide Gradient Gel Electrophoresis

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e. g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%)and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickett-sii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.