决明子降脂蛋白质的初步鉴定

目的　对具有降脂作用的决明子蛋白质进行初步鉴定。方法　用十二烷基硫酸钠 -聚丙烯酰胺凝胶电泳(SDS- PAGE)及毛细管等电聚焦 (CIEF)测定决明子降脂蛋白质的分子量和等电点。结果　SDS- PAGE结果表明 ,降脂蛋白质由分子量分别为 4 2 0 0 0 ,4 0 0 0 0和 380 0 0的 3种蛋白质组成 ;CIEF也表明 ,降脂蛋白质由等电点分别为 5 .88,5 .6 1和 1.95的 3种蛋白质组成。结论　降脂蛋白质为非均一蛋白质 ,尚需以降脂活性评价为导向 ,进行进一步的分离。     

浙江蝮蛇(Agkistrodon halys Pallas)蛇毒蛋白水解酶的分离纯化及其理化性质研究

应用DEAE-Sephadex A-50,Sephadex G-75,Sephadex G-200和QAE-SephadexA-25柱层析,从浙江蝮蛇毒中分离纯化出一种具有酪蛋白水解活性的蛋白水解酶a,称之为蛋白酶a.纯化后的蛋白酶经聚丙烯酰胺凝胶电泳和等电聚焦电泳鉴定,均呈现一条区带。等电点为6.4.用凝胶过滤及SDS-聚丙烯酰胺凝胶电泳测得其分子量为68000,糖蛋白染色法表明蛋白酶a是一糖蛋白。紫外吸收光谱表明蛋白酶a在277nm波长处有最大吸收,消光系数E_(277nm)~(0.1%)=0.681.蛋白酶a含有丰富的天冬氨酸、谷氨酸和赖氨酸。     

提取方法对“前愈”方中蜈蚣总蛋白质含量及凝胶电泳特征谱带的影响

目的:研究不同提取方法对"前愈"方中动物药材蜈蚣蛋白质含量及其凝胶电泳特征谱带的影响。方法:分别应用紫外可见分光光度计测定提取液中总蛋白含量;应用聚丙烯酰胺凝胶电泳技术获得提取液总蛋白凝胶电泳谱带,利用t检验分析试验结果。结果:水煎法及超声法提取蜈蚣30 min其蛋白质含量均高于15 min及45 min,且超声法各时间点提取量均约为水煎法的2倍;前愈复方合煎供试液其蛋白质含量要高于后下15 min及后下30 min供试液。蜈蚣的蛋白质分布范围在10～100 kDa。蜈蚣药材超声法提取液的蛋白质凝胶电泳特征谱带灰度值要明显高于相应时间点的水煎提取值。"前愈"复方中,合煎供试液其凝胶电泳灰度值最大。结论:提取方法对"前愈"方中动物药蜈蚣总蛋白质含量及凝胶电泳谱带影响较大,提示在后期"前愈"制剂工艺研究中应予以重视。     

液-质联用鉴别中药狼毒药材的来源

目的建立液-质联用鉴别中药狼毒药材来源的方法。方法瑞香狼毒、狼毒大戟及月腺大戟药材饮片分别粉碎后用70%乙醇超声提取30 min,提取液经微孔滤膜过滤后,利用液-质联用法进行分析。结果从3种狼毒药材提取物的液相色谱及质谱图中选择18个紫外吸收及质谱响应较好的色谱峰,并对其定性,可作为不同种属狼毒药材鉴别的特征峰。结论该方法简便快速,能够用于狼毒药材饮片来源的鉴别分析。     

天山假狼毒和瑞香狼毒根中挥发性成分研究

目的用GC-MS法分析比较天山假狼毒和瑞香狼毒根中挥发性成分的异同。方法采用索氏提取法提取天山假狼毒和瑞香狼毒根中的挥发性成分,经GC-MS联用仪对挥发性成分进行分析鉴定,采用色谱峰面积归一化法计算各挥发性成分的含量。结果共鉴定出2种植物根中的113个挥发性成分,其中天山假狼毒81个,瑞香狼毒69个,二者有37个共有成分。相对百分含量较高的成分有:十六酸甲酯(hexadecanoic acid-methyl ester)、9,12-顺式十八碳二烯酸[(Z,Z)-9,12-octadecadienoic acid]、角鲨烯(squalene)、1-庚三醇(1-heptatriacotanol)和异柠檬酸乙酯(ethyl iso-allocholate)。结论天山假狼毒和瑞香狼毒根中的挥发性成分有部分相近,但在含量与其他单一组分组成上存在一定的差异,本研究为天山假狼毒和瑞香狼毒药材的质量控制和进一步研究提供了重要理论依据。     

60例肾脏病人的尿蛋白盘状电泳分析

现将60例肾脏病人的尿蛋白盘状电泳分析结果报道如下。基本原理丙烯酰胺和甲叉双丙烯酰胺在催化剂作用下聚合成三维网状结构的凝胶,具有分子筛、浓缩和电荷效应。用十二烷基硫酸钠(SDS)与尿中蛋白质反应,形成带负电的SDS-蛋白质复合物,消除了蛋白质分子间原有的电荷差异。电泳时各种蛋白质都向正极移动,通过聚丙烯酰胺的浓缩效应和分子筛作用,分子量小的迁移较快,分子量大的迁移较慢,从而将蛋白质按分子量大小分离开来。材料与方法根据莽克强及陈梅芳等的方法略加修改、简化。尿蛋白定量用三氯醋酸法,上样量一律用200微克,标准蛋白用牛血清白蛋白。     

鹅不食草植物蛋白的纯化与含量测定

目的提取、纯化鹅不食草植物蛋白,并测定其含量。方法采用硫酸铵沉降法提取鹅不食草植物粗蛋白,通过透析、离子交换层析、分子筛层析等方法分离纯化所得粗蛋白。同时利用SDS-聚丙烯酰胺凝胶电泳法分析蛋白纯化情况,并应用Lowry法测定其含量。结果纯化后的鹅不食草蛋白电泳图谱条带多、清晰,Lowry法测定其蛋白含量高达85%以上。结论鹅不食草植物蛋白的纯化与含量测定方法简便、快捷、准确。为开发利用鹅不食草这一丰富的药用植物资源提供科学依据。     

瑞香狼毒化学成分研究(Ⅴ)挥发油化学成分研究

目的 通过对瑞香狼毒的化学组成进行分离,分别鉴定其化学成分.方法 所选取的方法为超声提取,乙醇提取,以及硅胶柱层析进行分析,通过IDNMR,2DNMR及MS方法来确定瑞香狼毒的组成结构.结果 分别得到5种化合物,通过对其光谱数据进行分析,确定这5种化合物分别为新瑞香素(I),-谷甾醇(II),伞形花内酯(III)、东莨菪素(IV)和胡萝卜苷(V).结论 其中为新天然产物的是化合物I,首次分离得到的为化合物3,即旋光纯的7-甲氧基狼毒素.     

眼镜蛇毒L-氨基酸氧化酶的纯化、性质鉴定及细胞毒性测定

目的从眼镜蛇蛇毒中分离L-氨基酸氧化酶（LAAO）,测定其理化性质及细胞毒性。方法采用Superdex75凝胶层析、Phenyl-SepharoseFF疏水层析、ResourceS离子交换层析分离纯化LAAO;SDS-聚丙烯酰胺凝胶电泳及C18反相色谱鉴定纯度并测定相对分子量;CCK8法测定细胞毒性。结果舟山眼镜蛇蛇毒经凝胶层析、疏水层析、离子交换层析及C18反相色谱后获得LAAO（暂定名NA-LAAO）,在非还原和还原条件下相对分子质量均为58kD左右;具有明显的抑制人胃癌MGC-803细胞增殖作用。结论从眼镜蛇毒中纯化得到LAAO,其具有明显的细胞毒性。     

梅花鹿鹿角托盘蛋白SDS-聚丙烯酰胺凝胶电泳指纹图谱研究

目的建立梅花鹿鹿角托盘蛋白SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）指纹图谱,为鹿托盘药材的质量评价提供新方法。方法采用SDS-PAGE法得到梅花鹿鹿角托盘蛋白的电泳胶片,通过凝胶电泳图像分析软件生成图谱后,经计算机辅助相似度评价系统进行评价并得到对照指纹图谱。结果梅花鹿鹿角托盘蛋白有6个特征带峰,14批样品指纹图谱相似度较高,方法稳定性、精确度、重现性良好。结论该方法实验简单易行、灵敏度高、专属性强,可用于梅花鹿鹿角托盘药材的质量评价。     

生脉注射液不同制备工艺中间体含残留蛋白质的检测研究

目的检测生脉注射液不同制备工艺中间体含残留蛋白质含量。方法以残留蛋白质为检测目标,采用透析法获取生脉注射液不同制备工艺中间体中大分子杂质,Bradford法测定各大分子杂质中残留蛋白质含量,结合SDS-PAGE电泳分析各残留蛋白质分子质量。结果在0～8μg线性范围内,牛血清白蛋白标准品回归方程为:Y=0.0474X-0.003(r=0.9974);不同工艺对提取液中残留蛋白的影响较大,红参不同工艺提取液含残留蛋白浓度较高,分别为399、353μg/mL;麦冬、五味子提取液残留蛋白含量相对较低;经对SDS-PAGE电泳结果分析,各不同工艺提取液中间体含蛋白质分子质量主要集中于6～11万。结论生脉注射液中间体提取工艺工程中可能有蛋白质残留,应加强残留蛋白质检测及生脉注射液的工艺改进;Bradford法结合SDS-PAGE电泳分析生脉注射液中的残留蛋白质,快速、准确、敏感性及检测结果均较好,可为工业生产提供科学数据参考。     

用SDS—填充胶毛细管电泳法测定蛋白质分子量

目的：建立蛋白质分子量测定的毛细管电泳方法。方法：以葡聚糖为分离介质,线性聚丙烯栈胺涂渍毛细管柱为支持介质,柱上２１４ｎｍ检测,进行蛋白质分离。结果：被分离的蛋白质分子量的对数与其相对迁移率之间具有良好的线性关系（ｒ＝０．９９７）;涂渍毛细管柱可以使用多达１００次面未出现分离效率的降低;同一毛细管柱和不同毛细管柱迁移时间的ＲＳＤ分别小于１．０％和１．３％;用十二烷基硫酸钠－毛细管胶电泳（ＳＤＳ－Ｃ     

Isolation and characterisation of a cysteine protease (phytolacain G), from Phytolacca americana roots

Protein extracts obtained from dried and fresh roots of Phytolacca americana L. (Phytolaccaceae) were examined in order to identify and characterise individual proteins. The extracts were compared with a commercial pokeweed mitogen standard using SDS polyacrylamide gel electrophoresis (SDS-PAGE). A dominant protein, present in both the extracts and the pokeweed mitogen standard, was isolated by subsequent ammonium sulphate fractionation, anion exchange chromatography, gel filtration and SDS-PAGE. In this way it was purified 140-fold with about 20 % yield and 70-fold with about 13 % yield from dried and fresh roots, respectively. Its molecular mass as determined by gel filtration and SDS-PAGE was estimated to be about 25 kDa. Subsequent isoelectric focussing revealed one single protein band at pH 6.0. LysC digestion of the 25 kDa protein yielded several peptides which were subjected to micro-sequencing. Comparison with published sequences revealed that the protein isolated was phytolacain G, a cysteine protease previously isolated from unripe fruits of P. americana L. The enzyme showed a high affinity towards the oxidised insulin B-chain and was completely inhibited by trans-epoxysuccinyl- L-leucylamido(4-guanidino)-butane (E64). The purified phytolacain G showed "lectin-like" activities such as haemagglutination and mitogenic effects towards human lymphocytes.     

绿豆胰蛋白酶抑制剂的分离和纯化

目的研究1种高效分离纯化绿豆胰蛋白酶抑制剂(MBTI)的方法。方法采用硫酸抽提,硫酸铵分级沉淀,缓冲液溶解沉淀,用相对分子质量(Mr)为30 000的超滤膜去除大分子蛋白质,Mr为5 000的超滤膜除盐及小分子蛋白质,亲和色谱纯化,得MBTI,高效液相色谱法(HPLC)和SDS-PAGE电泳法分别测定其纯度和Mr,以酪蛋白为底物测其活性。结果纯化的MBTI经HPLC和SDS-PAGE鉴定为2条蛋白质带,其Mr分别为12 000和8 000,其活性约为大豆蛋白酶抑制剂的3倍。结论超滤法与亲和色谱结合能快速高效地分离纯化MBTI。     

Separation of the components of type A botulinum neurotoxin complex by electrophoresis.

Clostridium botulinum neurotoxins (BoNTs) are the most toxic substances known. They exert potent neuroparalysis on vertebrates. C. botulinum produces seven serotypes of neurotoxin (A-G). BoNT/A, found in bacterial cultures of C. botulinum type A, is produced as a complex with a group of neurotoxin associated proteins (NAPs). Botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against the acidity and proteases of the stomach. Here, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation and identification of the constituent proteins of BoNT/A complex. A range of homogenous and gradient SDS-PAGE gels was used to resolve the BoNT/A complex. These gels were run under constant voltage and constant current conditions. The molecular weight and relative amount of each protein band were determined. On a 12.5% homogenous SDS-PAGE under reducing conditions, seven protein bands were identified with average molecular weights of 118, 106, 90, 56, 36, 23 and 17 kDa. The relative amounts of these seven proteins were determined densitometrically as 10, 6, 13, 27, 22, 13 and 8%, respectively. The separation and identification of BoNT/A complex will help in understanding the molecular structure and function of BoNT/A NAPs and their interaction with the toxin, in the toxico-infection process of the botulism diseased state. In particular, the stoichiometry of the individual components is established for a typical preparation of BoNT/A complex. Furthermore, the studies reported here identify the most favorable conditions for the baseline resolution of BoNT/A NAPs proteins for other workers in this field.     

蚯蚓新型脱氧核糖核酸酶的相对分子质量测定

目的测定蚯蚓组织中一种新型脱氧核糖核酸酶(EWD3)的相对分子质量。方法采用凝胶过滤层析法、十二烷基磺酸钠-聚丙烯酰氨凝胶电泳法(SDS-PAGE)和基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF-MS)3种方法测定一种新型蚯蚓脱氧核糖核酸酶(EWD3)的相对分子质量。结果凝胶过滤层析法、SDS-PAGE法和质谱法测定其相对分子质量分别为55080、58270和63460。结论凝胶过滤层析法和SDS-PAGE法可以常规粗略测定蛋白的相对分子质量,质谱法可以比较准确测定蛋白的相对分子质量,EWD3为单亚基蛋白,其相对分子质量约在55000￣65000之间。     

Purification and partial characterization of paralytic shellfish poison-binding protein from Acanthocardia tuberculatum.

A paralytic shellfish poison-binding protein (PSPBP) was purified 16.6-fold from the foot of the Moroccan cockles Acanthocardia tuberculatum. Using affinity chromatography, 2.5mg of PSPBP showing homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from 93 mg of crude extract. The purified PSPBP exhibits a specific activity of about 2.78 mU/mg proteins and has estimated molecular weight of 181 kDa. Observation of a single band equivalent to 88 kDa on SDS-PAGE under reducing conditions suggested it to be a homodimer. The optimal temperature and pH for the purified PSPBP were respectively 30 degrees C and 7.0.     

纳豆激酶的分离纯化及酶学性质研究

目的研究纳豆激酶分离纯化工艺及酶学性质。方法纳豆激酶发酵液的粗提物经Superdex 75凝胶色谱和聚丙烯酰胺凝胶电泳(PAGE)分离纯化,采用TAME法测定酶的活性,通过SDS-PAGE对纯化结果进行了检验。结果SDS-PAGE中显示单一色带,相对分子质量28000,以TAME为底物时纳豆激酶的米氏常数(Km)为35.47mmol/L,最适宜的温度37℃,最适宜pH为8.6。结论该分离纯化方法可以得到较纯的纳豆激酶。     

胸腺素α1-胸腺五肽在大肠杆菌中的表达及表达条件优化

目的探讨胸腺素α1-胸腺五肽(Tα1-TP5)在大肠杆菌中的表达及表达条件优化。方法将化学合成的Tα1-TP5基因与原核表达载体pGEX-4T-1融合并转化至E.coliBL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统研究培养基组成、诱导时机、诱导温度、诱导剂浓度及诱导时间等条件对融合蛋白表达量的影响。融合蛋白经GST琼脂糖珠亲和色谱纯化及重组肠激酶切割后,电喷雾质谱鉴定Tα1-TP5。结果选用TB培养基,在菌体对数生长中后期加入终浓度为0.05 mmol/L的IPTG,37℃诱导5 h,GST融合蛋白的表达量最高,占菌体总蛋白的35.8%,且主要以可溶形式表达。Tα1-TP5的分子质量与理论值相似。结论 Tα1-TP5成功在E.coli中表达,并确定了最佳表达条件。     

A low molecular weight protein with antimicrobial activity in the cutaneous 'venom' of the yellow-bellied toad (Bombina variegata pachypus)

The cutaneous 'venom' was collected from dorsal skin fragments of the yellow-bellied toad Bombina variegata pachypus by means of stimulation with noradrenaline. Light and electron microscope observations gave evidence that the 'venom' corresponds to the secretory products of both serous gland types (i.e. with small or large granules) characteristic of this genus, which had discharged their contents upon stimulation. The serous 'venom', when tested for antimicrobial activity, inhibited the growth of several bacterial strains. Heat treatment, dialysis, protease digestion and SDS-PAGE electrophoresis showed that the antimicrobial activity was thermostable and associated with a low molecular weight protein. This protein was purified and homogeneity determined by CM-cellulose chromatography and SDS-PAGE electrophoresis. The purified protein has a molecular weight of 6700, displays antibacterial properties and appears different from the antimicrobially active peptides previously isolated from the 'venom' of the toad.