利用色谱和飞行时间质谱方法分离并鉴定七鳃鳗血清中凝集素

本研究利用阳离子交换和分子筛的方法成功分离鉴定出分子量为105kDa,以多聚体形式存在七鳃鳗凝集素。实验结果表明,首先运用阳离子交换色谱方法能够将七鳃鳗血清混合组分进行初步分离,结合凝集活性鉴定方法,鉴定凝集活性最强组分存在于第二峰中。其次;利用分子筛方法将具有凝集活性组分进一步分离,最终得到分子量为105kDa左右具有凝集活性组分。结合Native-PAGE和SDS-PAGE结果表明,凝集活性组分以三聚体形式存在。同时,飞行时间质谱方法鉴定结果为凝集素（gi:13094239）。最终,体外鉴定结果表明七鳃鳗凝集素对兔红细胞和山羊红细胞均具有较强的凝集活性。七鳃鳗凝集素的分离纯化对研究七鳃鳗先天免疫防御机制和可变淋巴受体介导的适应性免疫防御机制均具有重要意义。     

病毒膜蛋白及其表达和纯化技术的研究进展

病毒囊膜是包膜病毒除核衣壳外的另一主要成分。它主要由多种蛋白质构成，称为膜蛋白，膜蛋白以单聚体，二聚体，三聚体，甚至更多的聚合体的形式存在，具有中和抗体，与靶细胞受体结合，凝集红细胞，诱导细胞融合等多种功能，膜蛋白可采用将其基因整合入载体，并在昆虫细胞，哺乳细胞或细菌中获得体外表达，膜蛋白可在去污剂的作用下形成蛋白质－脂－去污剂的复合物而被溶解，其中去污剂的临界微团浓度，亲水物－亲脂物平衡值，荷电、紫外穿透性等是选择合适去污剂的主要参考因子，溶解后的膜蛋白多利用聚丙烯酰胺凝胶电泳、凝胶过滤层析，离子交换层析，亲和层析，高效液相层析等技术并结合梯度离心；分级沉淀等进行分离、纯化。     

鲨鱼和魟鱼肝铁蛋白电泳纯的制备技术

采用硫酸铵分级盐析、DEAE阴离子交换层析、Sephacryl S—300凝胶层析技术分离纯化鲨鱼肝铁蛋白(Liver ferritin of Sphyma zygaena,SZLF)和虹鱼肝铁蛋白(Liver ferritin of Dasyatis akajei,DALF)。电子显微镜技术揭示了SZLF和DALF的分子结构,阐明了两者蛋白均由蛋白壳和铁核组成。柱层析和电泳技术拓展了电泳纯的SZLF和DALF制备方法,并可用于制备其他高纯度的铁蛋白。     

黄鳍金枪鱼酶解物免疫活性及其氨基酸分析

本文旨在研究黄鳍金枪鱼(Thunnus albacares)碎肉中4种不同蛋白酶酶解物的3k Da超滤组分对RAW264.7细胞的免疫活性。以RAW264.7细胞为实验模型, 采用噻唑蓝比色法检测细胞增殖活性, 中性红吞噬实验检测细胞吞噬能力, Griess试剂检测细胞培养上清液中NO含量, 并分析酶解物氨基酸组成和分子量分布。实验结果表明, 胰蛋白酶酶解物在12.5~200μg·mL^-1范围内无明显细胞毒性, 能增强RAW264.7细胞吞噬能力, 浓度为400μg·mL^-1时达到最大值132.50%(P<0.01), 浓度在50~200μg·mL^-1范围内能促进巨噬细胞NO释放(P<0.01), 且NO释放量与酶解液浓度存在一定的量效关系; 胰蛋白酶酶解物中必需氨基酸(essential amino acid, EAA)含量最高, 为51.09%, 其碱性氨基酸组氨酸、赖氨酸和精氨酸的含量分别为11.59%、11.00%和6.90%, 均高于其他蛋白酶酶解物, 并且分子量小于1k Da的小肽含量高达97.48%。研究结果表明, 4种蛋白酶酶解物中, 黄鳍金枪鱼碎肉胰蛋白酶酶解物的3k Da组分具有较明显的免疫活性, 这为其作为免疫活性肽的开发利用提供了理论依据。     

Advanced characterization of marine dissolved organic matter by combining reversed-phase liquid chromatography and FT-ICR-MS

Marine dissolved organic matter (DOM) was separated by reversed-phase (RP) liquid chromatography method and analyzed with fluorescence/absorption detection and Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS). The three key characteristics of the RP method are: (a) The C18-RP column chosen provides enhanced separation when the aqueous phase is 100% buffer-free water, and it does not degrade over time; (b) the water eluent adjusted to pH 7 significantly improves the resolution of water soluble compounds; (c) the initial flow maintained at low levels improves the separation of polar compounds. In samples, containing “fresh” DOM, specific peaks were detected, which were absent in “old” DOM samples. The combination with size exclusion chromatography (SEC) also demonstrated the relation between polarity and molecular size of DOM. FT-ICR-MS was applied to evaluate the quality of separation on a molecular scale demonstrating that physico-chemical characteristics of DOM can be related to molecular formulas. Sample extracts were separated into 4 preparative fractions, and a large suite of the identified molecular formulas only occurred in specific fractions. This is an important basis for the application of further analytical techniques in order to perform a more target-oriented analysis aiming at the determination of source and process biomarkers for DOM.     

羊栖菜提取物抗动植物病原菌活性及化学成分初步分析

采用纸片法对羊栖菜Sargassum fusiforme脂类的不同极性组分和乙醇提取物的薄层层析(TLC)分离带进行抗动植物病原菌的实验,结果表明,羊栖菜脂溶性化合物以弱极性脂为主,但其强极性脂具有较强的抗玉米大斑病菌活性.乙醇提取物有4条TLC分离带,分别表现出不同程度的抗菌活性,其中Rf=0.16带对肠炎病病原菌的抑制作用最强,抑菌圈达12mm.GC-MS分析羊栖菜的薄层层析分离带的组分发现多以醇类为主,15-冠-5醚可能是其高活性的来源.     

海洋真菌Neosartorya fischeri 1008F1的次级代谢产物抑菌及抗肿瘤活性的研究

通过抑菌及抗肿瘤活性实验,明确海洋真菌Neosartorya fischeri 1008F1次级代谢产物中的活性有效组分。应用对峙培养法和生长速率法,对菌株的抑菌活性进行测定。应用MTT法,对菌株的抗肿瘤活性进行测定。应用凝胶(Sephadex LH-20)柱层析的方法,对菌株发酵产物的提取物进行分离。结果发现,海洋真菌Neosartorya fischeri 1008F1的水溶性提取物对番茄早疫菌(Alternaria solani)具有一定的抑制活性。菌株的粗提物浸膏经Sephadex LH-20柱梯度洗脱后,得到的组分B、C、D、E和F。在0.5 g/L的供试浓度下,组分B、C和D对胃癌细胞SGC-7901的抑制率均高于80%,而组分B、C、D、E和F对肝癌细胞BEL-7404的抑制率均高于70%。     

Molecular masses and chromophoric properties of dissolved organic ligands for copper(II) in oceanic water

The distribution of molecular masses of organic ligands for copper(II) in oceanic water was investigated. The bulk dissolved organic matter (DOM) was fractionated by ultrafiltration and organic ligands were extracted from the resultant fractions by using immobilized metal ion affinity chromatography (IMAC). Contributions of total organic ligands were 2.0–4.4% of the bulk DOM in surface waters, as determined by the UV absorbance. In the distribution of molecular masses of organic ligands, relative contribution of the fraction with low molecular masses (<1000 Da) was dominant (49–62%), while 26–33% of the total organic ligands was in the 1000–10,000 Da fraction, leaving 10–19% in the >10,000 Da fraction. The distribution of molecular masses of organic ligands shifted to higher molecular masses, as compared with that of the bulk DOM. The fluorescence intensities of organic ligands were shown to be associated with carboxyl contents, based on peak excitation/emission wavelengths and the pH-dependence of fluorescence. Two ligand classes with different conditional stability constants (log K_CuL′≈7 and 9) were determined from fluorescence quenching of ligand fractions during copper(II) titration. Organic ligands in low molecular mass fractions were relatively weak and strong ligands occurred in higher molecular mass fractions. It is suggested that the weaker ligand sites would consist of two or more carboxyl groups (log K_HL′=4), whereas carboxyl groups (log =2), which are protonated at lower pH, and primary amine may additionally contribute to the formation of more stable copper(II) complexes of the stronger ligand.     

大米草中生物活性物质的筛选

大米草(Spartina anglica)是一种资源丰富的盐沼植物，目前已在中国沿海大量分布，综合开发利用大米草具有重要的生态意义和经济效益。利用硅胶柱层析方法分离大米草甲醇提取物，再分别用DPPH法、纸碟法和MTT法对大米草中40多个组分进行了抗氧化、抗细菌和抗肿瘤活性的筛选。结果得到抗氧化活性组分5个、抗菌活性组分2个、抗肿瘤活性组分3个。目前对其活性先导化合物的分离纯化研究正在进行中。     

近海温泉中嗜热菌Geobacillus sp. ZH1锰超氧化物歧化酶的克隆与表达

将嗜热菌Geobacillus sp. ZH1的超氧化物歧化酶(supseroxide dismutase,SOD)基因插入表达载体pET-32α(+),在Escherichia coli BL21(DE3)中进行表达,并利用亲和层析纯化重组超氧化物歧化酶.将制备的脱辅SOD进行Mn2+和Fe2+金属重构后,得到的Mn2+重构SOD的比活力达668U/mg,Fe2+重构Fe-SOD没有活性,说明ZH1菌株的超氧化物歧化酶为Mn-SOD.凝胶过滤及SDS-PAGE分析显示,Mn2+重构SOD为同聚二聚体,亚基分子量为71.7 kDa.这些研究结果为进一步研究该酶的生化特性及酶的定向进化研究奠定了良好的基础.     

Chemical studies of dissolved carbohydrates in seawater

Charcoal column chromatography and a dialysis system using a Visking tube were used for the concentration and separation of dissolved carbohydrates in seawater. Recoveries of mono-, oligo-, and polysaccharides were 46–74, 61–89 and 70%, respectively, relative to the total amount of sugars initially submitted to analysis. The combined charcoal column chromatography and dialysis system was used to separate dissolved carbohydrates in seawater samples from Mikawa Bay into mono-, oligo- and polysaccharide fractions. The polysaccharides are separated into two fractions with molecular weights of <4,000 and >4,000 using Sephadex G-25 column chromatography. Monosaccharides, oligosaccharides, polysaccharides with M.W.<4,000 and polysaccharides with M.W. >4,000 accounted for 7–9, 15–26, 7–13 and 20–33% of total dissolved carbohydrates, respectively. However, 32–38% of dissolved carbohydrates were lost during the analytical procedures. Glucose, galactose, mannose, xylose, arabinose, ribose, fucose, and rhamnose were found upon acid hydrolysis of the oligo- and polysaccharide fractions, while fucose and rhamnose were not present in the monosaccharide fraction. Possible sources of the carbohydrates in these fractions are discussed with reference to the monosaccharide composition of cellular carbohydrate in marine algae.     

仿刺参水溶性海参皂苷的分离制备及抗真菌活性的研究

利用海洋生物资源进行天然抗真菌新药的开发,从冻干海参加工废液中提取了水溶性海参皂苷,并对其抗真菌活性进行了研究。采用大孔吸附树脂法提取水溶性海参皂苷,经Libermann Burchard反应等对提取物的性质进行鉴定后,利用管碟法对所得提取物的抗菌活性进行了测定,并利用琼脂稀释法测定了所得水溶性海参皂苷对6株供试真菌的最低抑菌浓度(MIC)。提取物在Molish反应、沉淀反应和泡沫实验中均为阳性,说明提取物中确含有皂苷类成分;在Libermann Burchard反应中,液体的颜色处在红色与紫色之间,显示提取物可能是1种三萜类皂苷。水溶性海参皂苷对6株供试真菌均具有显著的抑制作用,且在浓度为0.5~4 mg/mL的范围内,其抑菌活性与所用浓度之间呈明显的正相关性;在浓度为4 mg/mL时,水溶性海参皂苷对6株供试真菌抑制活性的大小依次为裂殖酵母菌、啤酒酵母菌、白色念珠菌、葡萄炭疽病原菌、黄瓜枯萎病原菌和黑曲霉,海参皂苷对6株真菌的MIC值依次分别为0.002,0.016,0.063,0.063,0.125,0.250 mg/mL。     

重组七鳃鳗PHB2蛋白的原核表达及纯化

为了研究PHB2蛋白的生物学活性及后续抗体的制备,作者以前期构建的p MD18-T-Lm-PHB2质粒为模板,PCR扩增Lm-PHB2基因全长CDS区,PCR产物经Hind III和Eco R I双酶切后连接至表达载体p ET-32a,构建重组质粒p ET-32a-Lm-PHB2。将鉴定正确的重组质粒转化入大肠杆菌(Escherichia coli)Rosetta Blue,经IPTG诱导表达后,表达产物通过SDS-PAGE和Western blotting进行检测,利用镍柱亲和层析纯化得到纯度较高的目的蛋白r Lm-PHB2。结果表明,经PCR、双酶切和测序鉴定,所构建的重组质粒p ET-32a-Lm-PHB2序列正确,表达产物存在于裂解菌液的上清和沉淀中,经SDS-PAGE和Western blotting证实在约47 ku处有目的蛋白r Lm-PHB2的表达条带。本研究构建了重组七鳃鳗(Lampetra japonica)PHB2蛋白的原核表达载体,并大量表达和纯化目的蛋白,为该蛋白后续的抗体制备及生物活性研究奠定了基础。     

鸭毛藻内生真菌Hypocreales sp.的化学成分研究

从采自大连近海的红藻鸭毛藻（Symphyocladia latiuscula）中分离到一株肉座菌目真菌（Hypocreales sp．）,对其发酵代谢产物的化学成分进行了研究。利用正相硅胶柱层析、葡聚糖凝胶Sephadex LH-20柱层析、制备薄层层析（PTLC）以及重结晶等分离手段,并通过一维、二维核磁共振技术、质谱技术等从该菌发酵液中分离鉴定了10个化舍物：双酚A（1）;邻羟基苯甲酸（2）;吲哚甲酸（3）;吲哚乙酸（4）;N-乙酰色胺（5）;（22E,24R）-麦角甾-7,9,22三烯-3β-醇（6）;过氧化麦角甾醇（7）;（22E,24R）-5α,6α-环氧麦角甾-8,22-二烯-3β,7α-二醇（8）;（22E,24R）-麦角甾-7,22-二烯-6β-甲氧基-3β,5α-二醇（9）;啤酒甾醇（10）。这些化合物均为首次从该菌中分离得到,其中化合物1为首次作为天然产物分离得到。     

去污剂对裙带菜色素-蛋白质复合物电泳分离效果及其性质的影响

于1998年3月在青岛市太平角采集裙带菜孢子体，通过破碎、离心制备类囊体膜，用去污剂SDS、DOC、DMG和DIG增溶裙带菜类囊体膜，经PAGE分离色素-蛋白质复合物，并测定其光谱特性，结果表明，电泳分离效果与所用的去污剂的种类有关。去污剂不同，分离到的同一类型复合物的吸收光谱有差异，捕光色素-蛋白质复合物的数目、种类及吸收光谱明显不同，电泳中产生的游离色素的多少也随去污剂种类而异，在4种去污剂中，以光谱明显不同，电泳中产生的游离色素的多少也随去污剂种类而异，在4种去污剂中，以DMG和DIG增溶的效果最好，经SDS-PAGE分出6种色素-蛋白质复合物，游离色素较少，DMG和DIG增溶的效果最好，经SDS-PAGE分出6种色素-蛋白质复合物，游离色素较少。     

4 种病原弧菌外膜蛋白的提取及抗原性初步分析

利用十二烷基磺酸钠抽提并结合超速离心的方法提取了鳗弧菌(Vibrio anguillarum)、河口弧菌(V.aestuarianus)、霍乱弧菌(V.cholerae)和副溶血弧菌(V.parahaemolyticus)4种致病性弧菌的主要外膜蛋白,通过SDS-PAGE分析比较4种弧菌主要外膜蛋白的组分。结果表明:4株弧菌的外膜蛋白电泳图谱一般可得到5～10条蛋白带,分子量多数集中在26~40 kD和48~85 kD。对所提取的4种弧菌主要外膜蛋白进行SDS-PAGE后,分别与自制的兔抗鳗弧菌血清、抗河口弧菌血清、抗副溶血弧菌血清进行Western-blotting印迹分析。结果显示每种全菌抗血清可与相应菌的外膜蛋白部分组分发生免疫反应,并与其他3种弧菌外膜蛋白的部分组分产生交叉免疫反应,这些反应条带分子量主要集中于30~48 kD之间。本研究为进一步研究致病性弧菌外膜蛋白免疫学特性提供参考。     

经酶修饰和未经酶修饰的鹌鹑红细胞对甲壳动物血清凝集素凝集活性的差异研究

为寻找凝集素作为分子探针提供基础资料,用12种甲壳动物血清抽提液对天然的或经酶修饰的鹌鹑红细胞进行凝集试验。实验结果表明,经蜗牛酶修饰后的鹌鹑红细胞的凝集作用发生了变化,与未经酶处理的鹌鹑红细胞相比,凝集活性提高的有日本沼虾（Macrobrachium nipponense）等7种,凝集活性下降的有罗氏沼虾（Macrobrachium rosenbergi）等2种。凝集素对动物红细胞的凝集可看作是与这些细胞表面的多糖或糖蛋白——即受体的识别性鲒合,这表明鹌鹑红细胞表面具有12种凝集素的受体,且大多数受体都受到酶修饰的影响,从而在凝集活性上表现出差异。     

单环刺螠纤溶酶的分离纯化及其性质的初步研究

从单环刺中制得单一组分的纤溶活性纤溶酶,并对其性质进行初步研究.单环刺体组织液经离心,超滤,离子交换层析,凝胶过滤等方法进行分离纯化,制得单一洗脱峰酶组分,经Native-PAGE电泳分析,SDS-PAGE电泳分析和飞行质谱分析,鉴定了酶制品纯度和相对分子量,进行了体外溶血栓试验.所得纯品UFEIa水解酪蛋白的比活力为442.0 U/mg,纯化倍数为12.1倍,回收率为27.0%.UFEIa为单一组分,相对分子量约23 800 Da.体外溶血栓实验表明此纤溶活性蛋白酶与蚓激酶相比具有很好的溶血栓活性.     

ISOLATION OF DNA FROM SEA URCHIN SPERM NUCLEI BY HYDROXYAPATITE CHROMATOGRAPHY

This article describes in detail the procedures in obtaining spermatids from living animals and in isolating sperm nuclei DNA by hydroxyapatite chromatography. The isolated DNA is free of RNAs and proteins after treatment with proteinase K, followed by chromatography on hydroxyapatite column which is eluted with stepwise-increased concentrations of potassium phosphate buffer. More than 89% of the DNA purified is found in the eluates of both 0.2 M and 0.25 M potassium phosphate buffer. The two fractions are identical in ultraviolet absorption spectrum. DNA molecules thus prepared should be useful for studying DNA structure and DNA replication by electron microscopy, since the length of the purified DNA molecules is in the range of the size of a replicating unit.     

栉孔扇贝(Chlamys farreri)中抗氧化肽的分离纯化及性质研究

以栉孔扇贝内脏团为原料,对其中抗氧化肽的制备、纯化及性质进行了研究。首先采用硫酸铵沉淀、DEAE-Sephadex A-50阴离子交换层析的方法,进行了扇贝提取蛋白的制备,其次采用海洋蛋白酶YS-80水解、SephadexG-25凝胶层析的方法,进行扇贝抗氧化肽的制备,获得多肽粗品(海洋肽,PCF),并进一步采用CM Sepharose阳离子交换层析和反相高效液相色谱的方法,对扇贝抗氧化肽进行了纯化,结果得到组分PCF-3A,反相高效液相显示为单一峰。采用茚三酮反应、Sephadex G-15凝胶层析和AccQ.Tag氨基酸分析柱,进行了PCF-3A的理化性质研究。结果表明,茚三酮反应呈阳性;分子量约为794D;由7种氨基酸组成,分别为天冬氨酸、甘氨酸、苏氨酸、丙氨酸、缬氨酸、赖氨酸和脯氨酸。采用邻苯三酚自氧化体系和Fenton反应体系对PCF-3A的抗氧化活性进行了研究,发现该肽可有效清除羟自由基和超氧阴离子,半抑制浓度(IC50)分别为0.39mg/ml和2.85mg/ml,具有明显的抗氧化、抗衰老作用。