Fusarium moniliforme secretes four endopolygalacturonases derived from a single gene product

Extracellular endopolygalacturonase, purified from the pathogenic fungus Fusarium moniliforme, consists of four molecular forms (38, 41·5, 45, and 48·5 kDa, respectively. Three forms (38, 41·5, and 45 kDa) were purified to homogencity by FPLC on a Mono S column followed by electroelution after SDS-PAGE. The N-terminal amino acid sequences of each of the three forms, and of a mixture containing all four forms were shown to be identical to that predicted from the nucleotide sequence of the endopolygalacturonase gene previously cloned from F. moniliforme. Enzymatic deglycosylation experiments revealed the presence of N-linked, high mannose oligosaccharide side-chains on all four forms of endo polygalacturonase. Hydrogen fluoride catalysed chemical deglycosylation of the polygalacturonase mixture yielded a single polypeptide with an apparent molecular mass of 36·2 kDa. Southern blot analysis, carried out at high stringency with an endopolygalacturonase-specific probe on genomic DNA digested with three different restriction enzymes, showed a single hybridizing restriction fragment in all three digests. A single 2·0 Mb chromosome hybridized with the endo polygalacturonase-specific probe, as shown by Southern blot analysis of F. moniliforme chromosomes separated by CHEF electrophoresis. Northern blot analysis revealed only one mRNA species 1350 nt encoding endo polygalacturonase. These data indicate that a single gene encodes the endopolygalacturonases of F. moniliforme.     

棉花黄萎病菌致萎毒素的分离纯化方法

利用快速蛋白质液相色谱技术（Fast Protein Liquid Chromatography,FPLC)，对棉花3个黄萎病菌（Verticillium dahliae)菌系外泌毒素的某些理化性质进行了分析，结果显示，黄萎病菌毒素有两个致萎峰，大分子峰I毒蛋白致萎力较弱，致萎症状属黄斑型，分子峰Ⅱ毒蛋白其致萎力极强，致萎症状属青枯型，透析试验表明，可透8000－10000Da的透析袋会使小分子峰Ⅱ丢失，热稳定试验表明，峰I，峰II毒蛋白皆可耐100℃高温水浴热煮而不发生沉淀，并保持致萎活性，根据以上研究结果，本文提出了黄萎病毒分离纯化新方法。     

快速蛋白液相色谱分离小麦高分子量谷蛋白亚基方法的研究

高分子量谷蛋白亚基作为小麦贮藏蛋白的重要组成部分,其组成和含量对小麦面粉的烘烤品质和加工品质有重要影响。本研究以普通小麦品种小偃22号为材料,采用快速蛋白液相色谱（Fast Protein Liquid Chromatograth,FPLC）凝胶过滤来分离高分子量谷蛋白亚基,并从样品浓度、洗脱液和流速3个方面进行了研究,发现将原样品稀释8倍、洗脱液为尿素、流速为0．3ml／min时洗脱效果最好。     

Bovine CD4+ T-cells lines reactive with soluble and membrane antigens of Cowdria ruminantium.

Cowdria-specific CD4⁺ T-cell lines generated from immunised cattle respond to both soluble and membrane proteins of the agent. Furthermore, the lines produced the Cowdria-inhibitory cytokine IFN-γ in response to soluble antigens fractionated by gel filtration and FPLC. Activity eluted as a single peak around fraction 15 for all T-cell lines tested. This fraction induced the highest production of IFN-γ by the lines and was shown by SDS-polyacrylamide gel electrophoresis and silver staining analysis to contain less than 10 different bands ranging from 22 to 32 kDa. Given their high sensitivity and specificity, these short-term CD4⁺ T-cell lines will be valuable tools for the identification of Cowdria antigens for incorporation in a subunit vaccine.     

克仑特罗偶联物的鉴定

克仑特罗经偶氮化后,与牛血清白蛋白在碱性条件下发生偶联反应生成偶联物.偶联物经红外光谱、紫外光谱、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳、快速蛋白液相色谱及免疫鉴定,确定偶联成功.对上述几种偶联物鉴定方法进行了简单比较,认为快速蛋白液相色谱是鉴定偶联物的最佳方法.     

新疆西瓜花叶病毒2号外壳蛋白的研究

 本文以马铃薯Y病毒组（potyvirus group）确定成员西瓜花叶病毒2号（WMV-2）为研究对象，对新疆和澳大利亚WMV-2的外壳蛋白制备、快速蛋白液相色谱（FPLC）肽图谱进行了研究和比较。通过分析两者外壳蛋白同源性的大小，证明新疆和澳大利亚WMV-2是同种马铃薯Y病毒的不同株系。从而说明外壳蛋白的FPLC肽图谱可成功地用于马铃薯Y病毒组的鉴定和分类。     

水稻白叶枯病菌解毒素蛋白提纯和作用机制研究

采用 (NH4) 2 SO4分步沉淀法 ,从水稻白叶枯病菌培养滤液中提取对水稻白叶枯病菌有机酸毒素具解毒作用的解毒蛋白粗提物 (crudedetoxificationprotein ,CDP) ,经滚动式等电聚焦电泳 (RPIE)和离子交换层析 (FPLC)分离 ,生物测定结果表明 ,第Ⅱ吸收峰具有生物活性 ;再经SDS PAGE电泳和考马斯亮蓝染色 ,第Ⅱ吸收峰收集物有明显的一条带 ,相对分子质量为 4 7 9× 10 3 。野生菌产生的CDP解毒活性明显低于其毒性基因突变体。CDP对热和蛋白酶K均不稳定 ,对毒素的解毒主要是通过脱羧起作用。用CDP处理水稻后 ,水稻体内与抗病性相关的酶活性显著提高 ;接种水稻白叶枯病菌 ,病斑长度明显降低     

Bovine CD4 T-cell lines reactive with soluble and membrane antigens of Cowdria ruminantium

Cowdria-specific CD4⁺ T-cell lines generated from immunised cattle respond to both soluble and membrane proteins of the agent. Furthermore, the lines produced the Cowdria-inhibitory cytokine IFN-γ in response to soluble antigens fractionated by gel filtration and FPLC. Activity eluted as a single peak around fraction 15 for all T-cell lines tested. This fraction induced the highest production of IFN-γ by the lines and was shown by SDS-polyacrylamide gel electrophoresis and silver staining analysis to contain less than 10 different bands ranging from 22 to 32 kDa. Given their high sensitivity and specificity, these short-term CD4⁺ T-cell lines will be valuable tools for the identification of Cowdria antigens for incorporation in a subunit vaccine.     

培养方式对蜜环菌胞内多糖合成与抗眩晕症活性影响的研究

对蜜环菌在不同液态培养方式下胞内多糖的合成、组成以及抗眩晕症活性进行比较。结果显示,蜜环菌在摇床培养条件下形成菌丝球,菌丝多糖(AMP)和生物量在第8d达到最高产量,分别为124.32mg/L和10.85g/L,蜜环菌在静置培养条件下形成菌索,菌索多糖(ARP)与生物量在20d时达到最高,分别为588.40mg/L和37.17g/L,静置培养对蜜环菌胞内多糖的合成有利;两种多糖在多糖含量、蛋白质含量、糖醛酸含量以及皂苷含量等组成成分上基本一致;AKTA-purifier快速层析纯化系统(FPLC)检测显示,AMP与ARP有四个相同的多糖组分,ARP另多出一个组分;抗机械旋转所致小鼠眩晕症试验表明AMP与ARP活性相似,均能显著缩短眩晕小鼠逃避电击所用的时间(P<0.01),增加小鼠眩晕后的进食量,因而对机械旋转所致眩晕症具有一定疗效。