二氧化碳噬纤维全菌蛋白SDS—PAGE技术鉴定的研究

本文对不同菌株二氧化碳噬纤维菌的全菌蛋白采取ＳＤＳ－ＰＡＧＥ结合考马斯亮兰染色和染色两种染色方法进行初步研究,结果表明二氧化碳碳噬菌临床分离株和参考菌株间各临床分株间泳图图形存在差异。     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝球脂糖亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ＫＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值。在以Ｇｌｕ为源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ     

地衣芽孢杆菌β—甘露聚糖酶的纯化及其性质的研究

地衣芽孢杆菌１Ｂａｃｉｕｕｓ Ｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＢＬ－３０６产生的胞外β－甘露聚糖酶经硫酸铵分级盐析,ＤＥＡＥ－纤维素柱层析。Ｓｅｐｈａｄｅｘ－Ｇ１００柱凝胶过滤和ＤＥＡＥ－纤维素柱再层析分离纯化,得到ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）均一样品。用ＳＤＳ－ＰＡＧＥ测得纯化后β－甘露聚糖酶分子量为２６０００道尔顿。用凝胶等电聚焦电泳（ＰＡＧＥＩＥＦ）测得等电点ＰＩ为５．０。该酶     

甲基单胞菌Methylomonassp.GYJ3中甲烷单加氧酶还原酶组分的…

Ｍｅｙｌｏｍｏｎａｓ ｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ阴离子交换层析,Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌ ＨＰＬＣ分离纯化出ＭＭＯ还原酶组分,经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白,ＳＤＳ－ＰＡＧＥ电泳表明的酶由     

神经营养因子GDNF编码顺序的克隆及表达产物的纯化

根据基因库中的顺序,设计了胶质细胞源神经营养因子（ＧＤＮＦ）基因的ＰＣＲ引物,以此从人基因组ＤＮＡ中扩增并克隆了ＧＤＮＦ的编码序列,经ＤＮＡ测序确认后,该片段克隆到表达质粒ｐＥＴ－３ａ中,转化大肠杆菌ＢＬ２１（ＤＥ３）．培养的重组菌经ＩＰＴＧ诱导,在Ｔ７启动子调控下表达出ｈＧＤＮＦ蛋白．经电泳分析表明ＧＤＮＦ主要存在于细菌包涵体中．从培养菌中制备包涵体,经充分洗涤,溶解于含８ｍｏｌ／Ｌ尿素的变性缓冲液中．经ＳＰ－Ｓｅｐｈａｒｏｓｅ柱层析分离,梯度洗脱,以１５％ＳＤＳ－ＰＡＧＥ检查含ＧＤＮＦ的部分．将含单体ＧＤＮＦ部分进行复性,再次用ＳＰ－Ｓｅｐｈａｒｏｓｅ离子柱分离同源二体ＧＤＮＦ．最后经ＳＤＳ－ＰＡＧＥ制备电泳纯化,纯度大于９５％．经Ｎ端测序表明序列正确．经测定,每升培养菌可得约１０ｍｇ纯化的ＧＤＮＦ．     

小麦高分子量麦谷蛋白亚基分离方法的研究

小麦高分子量麦谷蛋白亚基（ＨＭＷ－ＧＳ）与小麦面包烘烤质量和面粉的加工特性密切相关,ＳＤＳ－ＰＡＧＥ是其常用的分离方法之一。ＳＤＳ－ＰＡＧＥ方法一般分为２类：第一类采用１１％和５％浓度的胶,后者用于分离２亚基和２＾＊亚基,该种方法常使用碱性提取液,需要２次电泳过程,且在５％浓度的胶中ＨＭＷ－ＧＳ易于和麦醇蛋白混淆;另外一类ＳＤＳ－ＰＡＧＥ采用梯度胶,配合使用银染方法,制梯度胶则使用梯度仪及磁力搅拌     

SDS－PAGE中蛋白质回收方法

ＳＤＳ－ＰＡＧＥ中蛋白质回收方法傅国平（南京经济学院食品科学与工程系,南京２１０００３）关键词蛋白质回收ＳＤＳ－ＰＡＧＥ作为一种常规的蛋白质分离及分析手段,具有简便、迅速的优点,但从凝胶中回收所需蛋白质往往比较困难,主要是由于经考马期亮蓝染色及固定后...     

诺卡氏菌形放线菌β-甘露聚糖酶的纯化和性质

产β－１,４－Ｄ甘露聚糖酶的诺卡氏菌形放线菌（Ｎｏｃａｒｄｉｏｆｏｒｍ ａｃｔｉｎｏｍｙｃｅｔｅｓ）菌株ＮＡ３－５４０,发酵培养７２ｈ,发酵液离心去菌体,上清经硫酸铵沉淀,９５％乙醇沉淀,ＣＭ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析、羟基磷灰石柱层析、ＤＥＡＥ－纤维素离子交换及Ｓｅｐｈａｄｅｘ Ｇ－１００分子凝胶过滤柱等步骤,β－甘露聚糖酶的比活提高了１３７倍,获得凝胶电泳均一的蛋白样品。经ＳＤＳ－ＰＡＧ     

高及低转移潜能小鼠肝癌细胞株糖蛋白凝集素结合特征的研究

为探讨肿瘤转移与细胞表面的糖结构的关系,对小鼠肝癌细胞的高、低淋巴道转移株ＨＣａ－Ｆ和ＨＣａ－Ｐ进行了蛋白质电泳及经蛋白质印迹术后的５种凝集素（ＣｏｎＡ、ＷＧＡ、ＵＥＡ、ＳＢＡ、ＰＮＡ）结合糖蛋白谱的对比分析。结果表明：高、低转移两株细胞的ＳＤＳ－ＰＡＧＥ谱基本相同;Ｃｏｎ Ａ特异结合糖蛋白共有５种（￣７２,８０￣９０,￣１０４,￣１５０,￣２００ｋＤ）;其中较明显的差异为￣７２ｋＤ ＣｏｎＡ特异     

荧光假单胞菌抗噬菌体菌株的选育

本实验从荧光假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｆｌｕｏｒｅｓｃｅｎｓ）ＡＳ—３菌株的不正常发酵液中分离到一种噬菌体,将其命名为ＰＦＡＳ。ＡＳ—３菌株能利用葡萄糖发酵产生Ｄ－异维生素Ｃ的前体物质２－酮基－Ｄ－葡萄糖酸。电镜观察表明ＰＦＡＳ噬菌体呈蝌蚪形,具有直径为６６ｎｍ的六角形头部及长１１７ｎｍ的尾部。通过紫外线诱变及自然选育两种途径,配合简便有效的初筛方法,经多次分离、纯化、复筛最终在摇并发酵试验中获得６株产量稳定地高于对照敏感菌的抗噬菌体菌株,可望用于生产。     

Rapid identification of medically important yeasts by electrophoretic protein patterns

A simple electrophoretic method for yeast identification was evaluated. Whole cells were extracted by SDS and the protein profiles obtained in SDS-PAGE after Coomassie blue staining were compared for 52 strains from 9 species of yeast or yeast-like fungi commonly isolated from man (Candida albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, C. tropicalis, Geotrichum candidum, Saccharomyces cerevisiae). The corresponding patterns showed 30 to 45 polypeptides in the range 95-20 kDa and were clearly different for the 9 species. No differences could be detected between strains from the same species. The characteristic patterns were obtained within 24 h allowing rapid identification of the most commonly encountered clinical yeast isolates.     

Restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal DNA of human Capnocytophaga

M.J. WILSON, W.G. WADE AND A.J. WEIGHTMAN. 1995. The confusion in the taxonomic status of the genus Capnocytophaga has made identification of strains and studies on the role of this genus in infectious diseases equivocal. In this study 33 strains of Capnocytophaga including reference strains and various clinical isolates, were studied using RFLP analysis of 16S ribosomal RNA genes. The 16S ribosomal RNA (rRNA) gene sequences from whole cell suspensions and isolated genomic DNA samples were amplified by the polymerase chain reaction (PCR) using eubacterial specific primers. PCR products were purified and characterized by single digestions with 12 restriction endonucleases. Five of these, BanI, CfoI, HaeIII, HphI and RsaII were found to discriminate reproducibly between strains, and restriction patterns (ribotypes) produced by these were analysed to clarify the classification of Capnocytophaga strains. Dendrograms inferring similarities were derived from these data by the UPGMA method. This analysis produced three major clusters of strains, each of which was associated with a previously proposed species type strain: C. gingivalis, C. sputigena and C. ochracea. The results support the division of Capnocytophaga into three species and demonstrate that, despite the heterogeneity of this genus, the modified ribotyping method provides a simple, rapid and reproducible way to identify Capnocytophaga strains.     

Application of multilocus enzyme electrophoresis in studies of the epidemiology of Listeria monocytogenes in Denmark.

A total of 245 strains of Listeria monocytogenes were investigated. These strains were isolated from human and animal cases of listeriosis as well as from different kinds of raw and processed foods. Thirty-three electrophoretic types (ETs) were identified among the 245 strains. The strains investigated included all human clinical strains isolated in Denmark during 1989 and 1990. Seventy-three percent of the strains isolated in this period were assigned to one of only two ETs (ET 1 and ET 4). ET 1, which was found to be the most frequently occurring ET among strains isolated from human clinical cases, was also found to occur rather frequently in animal clinical cases. ET 1 was, however, found only sporadically among strains isolated from foods and food factories. The data indicate that there might be something distinctive about the physiology or ecology of the ET 1 clone which makes it more likely to bring about disease in human beings either because of high pathogenicity or because of a special ability to multiply to infectious doses in processed foods. Another type, designated ET 4, was found to be the next most frequently occurring ET, after ET 1, among human clinical isolates. This could be explained by the fact that ET 4 was found to be the most frequently occurring ET within food isolates.     

A polyphasic taxonomic study of Chryseobacterium strains isolated from dairy sources

A polyphasic taxonomic study, employing protein electrophoresis (SDS-PAGE), gas chromatographic analysis of cellular fatty acids (FAME), mol% G+C determination and DNA-DNA hybridizations, was undertaken on 103 dairy isolates shown to belong to Chryseobacterium. Reference strains of the Chryseobacterium species, CDC group IIb and Embedobacter brevis were included. SDS-PAGE analysis yielded good differentiation between the investigated species. About half of the strains could be clustered into nine major groups while the other half occupied a separate position. With FAME analysis no clear differentiation of the Chryseobacterium species (except C. meningosepticum) and SDS-PAGE groups could be achieved. FAME analysis, however, gave good differentiation between the Chryseobacterium and Empedobacter strains. The mol% G+C of the isolates tested, ranged between 36.4 and 39.0. The combination of SDS-PAGE and DNA-DNA hybridization identified a large group of dairy isolates as C. indologenes, one isolate as C. gleum and two new genotypic groups, comprising five and 15 dairy isolates respectively, emerged from the polyphasic study. Another large part of strains have a separate or uncertain position in Chryseobacterium and remained classified as Chryseobacterium species CDC group IIb.     

Genetic heterogeneity among Vibrio alginolyticus isolated from shrimp farms by PCR fingerprinting

AIMS: To study the strain variability among Vibrio alginolyticus isolates from different sources by insertion sequence-targeted PCR fingerprinting and whole cell protein profile analysis. METHODS AND RESULTS: Eleven strains of V. alginolyticus were isolated from seven different sources including healthy, infected, farm-reared and wild shrimps. Following biochemical characterization, the isolates were analysed by PCR fingerprinting and whole cell protein analysis by SDS-PAGE. The strains were genetically different irrespective of the sources of isolation. CONCLUSIONS: Strain variation exists in V. alginolyticus isolates obtained even from the same source, and PCR fingerprinting is a simple and efficient method in identifying strain-specific variations among the different isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio alginolyticus is implicated in severe vibriosis of marine aquaculture systems although many strains are avirulent and could be used as probiotic strains. As a wide variation exists among this species, differentiating the harmful and beneficial strains would help in finding ways of controlling the infections by eliminating harmful shrimp pathogenic vibrios.     

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.     

A rapid method for preparation of rough and smooth lipopolysaccharide from Bacteroides, Porphyromonas and Prevotella

Abstract We describe a new method for lipopolysaccharide (LPS) preparation by water extraction at 100°C and subsequent digestion with proteinase K. The crude LPS could be reliably used for immunoblotting since it retained a high level of antigenicity, and was free of SDS and proteinase K, both of which can cause problems. Two monoclonal antibodies which failed to react with LPS prepared by two conventional methods reacted well with our preparation. We used the new method to prepare LPS from 44 strains of bacteria formerly classified as Bacteroides , some of which have been reclassified as Porphyromonas or Prevotella . In general, yields were good, and electrophoretic profiles obtained with SDS-PAGE and silver staining enabled strains to be rated rough, semi-rough, or smooth.     

Variations in virulence between different electrophoretic types of Listeria monocytogenes

A total of 245 strains of Listeria monocytogenes, representing 33 different electrophoretic types (ETs), were examined quantitatively for haemolytic activity. No significant difference was observed in the mean haemolytic activity between different ETs. Eighty four out of 91 strains examined were found to be virulent for chick embryos. Strains belonging to ET 2 and ET 4 were found to be less virulent than strains of other ETs (P = 0.0447). Furthermore, strains from clinical cases were found to be more virulent (P = 0.0002) than strains from foods (the MTD among clinical strains was 2.46 in mean compared with 3.64 among food isolates). The explanation for this may be that more virulent strains are more prone to cause human infection. It is, however, also possible that strains of L. monocytogenes may become more virulent while multiplying in a living organism compared with multiplying in foods.     

INTRASPECIFIC VARIATION IN THE DINOFLAGELLATE GAMBIERDIZSCUS TOXICUS (DINOPHYCEAE). I. ISOZYME ANALYSIS

Intraspecific variation among 19 isolates of the ciguatera-causing dinoflagellate Gambierdiscus toxicus Adachi & Fukuyo (Dinophyceae) collected from French Polynesia, New Caledonia, and the French West Indies was investigated by isozyme analysis. Comparison of their cell sizes and growth rates revealed that significant variation exists among these clones. Comparison of electrophoretic patterns for seven enzyme systems indicated that G. toxicus is comprised of numerous biochemically distinct strains. Isolates from Tubuai and Hao appeared to be the most distantly related. Tahitian strains of G. toxicus also showed a remarkably low degree of similarity with the Tubuai isolates. The latter, which were taken from the same locale in Tubuai, also exhibited highly heterogeneous electrophoretic Profiles when compared to each other, suggesting a multiclonal origin. The single isolate analyzed from the Atlantic Ocean was most closely related to Tahitian isolates, despite their geographic separation. Finally, no clear relationship was found between the electrophoretic profiles of these isolates and their capacity to produce ciguatoxic compounds .     

Electrophoretic patterns of lipopolysaccharides and antigenic analysis of soybean bradyrhizobia

Several serological methods have been used for the characterization and identification of soybean bradyrhizobia. However, some problem were non-reactivity of certain strains and cross-reactivity among others. Since lipopolysaccharide (LPS) can often be used in strain identification, the objective was to investigate the antigenic properties and polyacrylamide gel electrophoretic pattern of 12 Brazilian strains of Bradyrhizobium japonicum that nodulate soybean and to compare them to standard strains. The close correlation between the LPS patterns obtained by SDS-PAGE and the serological analysis permitted us to assign the strains to nine groups different or the same as the standard strains.