一株番茄灰霉病菌拮抗菌的系统鉴定

Y-21是从山东省淄博市蔬菜温室中分离到的一株细菌,通过室内平板对峙试验,对番茄灰霉病菌具有较强的拮抗性。本研究通过形态观察、生理生化反应和分子生物学方法对这株细菌进行系统鉴定。显微镜观察菌株形态为杆状;有芽孢;革兰氏染色呈阳性。采用经典的基因组提取方法提取基因组DNA,以其为模板,利用引物63F、1494R扩增16SrRNA基因,回收16SrRNA基因的PCR产物并与T载体连接,通过热激法将重组质粒转人大肠杆菌感受态细胞,利用蓝白斑筛选,挑出白斑,提取质粒,通过蓝白斑质粒的电泳图比对和质粒的PCR检测确定阳性克隆,将阳性克隆菌株送样测序。将测序结果输入BLAST进行比对,发现Y-21属于假单胞菌属,然后对其构建系统发育树。     

一株絮凝剂产生菌的16s rRNA鉴定和絮凝性能研究

通过直接测序PCR产物的方法,获得了絮凝剂产生菌Lv1-2的16s rRNA基因序列,通过BLAST比对和系统进化树分析,将该菌鉴定为胶质芽孢杆菌（B．mucilaginosus）。对该菌产生的絮凝剂的热稳定性和助凝剂CaCl2的投入量进行了研究。结果表明,CaCl2有较好的促进絮凝的作用,絮凝率可提高10％;在30℃-90℃范围内絮凝剂有较好的热稳定性。     

In-Fusion克隆技术构建HBVX基因真核表达质粒

目的以血清中乙型肝炎病毒（HBV）DNA 为模板,克隆构建HBV X 蛋白质的真核表达载体pcDNA-HBx.方法：设计扩增HBV X 基因的In-Fusion PCR 引物,采用高保真DNA 聚合酶分两段扩增HBV X 基因序列;采用In-Fusion 克隆技术,将两段X 基因扩增片段-步克隆入经Hind Ⅲ 和Xba I 双酶切的pcDNA3.0 真核表达载体,以构建重组真核表达载体pcDNA-HBX ;采用Hind Ⅲ 和Xba I 双酶切和DNA 序列测序筛选鉴定重组载体;pcDNAHBx转染HepG2 细胞,Western blot 检测pcDNA-HBx 转染细胞的HBx 蛋白质表达.结果：In-Fusion PCR 引物分两段扩增获得全长HBx 基因序列;In-Fusion 获得克隆的pcDNA-HBx 经双酶切筛选得到阳性重组质粒,测序分析证实插入序列正确;转染pcDNA-HBX 的HepG2 细胞,Western blot 证实表达HBx 蛋白质.结论：以血清HBV DNA 为模板克隆HBV X 基因,构建了表达HBV HBx 蛋白质的真核表达载体,为HBx 蛋白质的生物学功能研究提供支持.     

In-Fusion克隆技术构建HBV X基因真核表达质粒

目的 :以血清中乙型肝炎病毒(HBV)DNA为模板,克隆构建HBV X蛋白质的真核表达载体pcDNA-HBx。方法 :设计扩增HBV X基因的In-Fusion PCR引物,采用高保真DNA聚合酶分两段扩增HBV X基因序列;采用In-Fusion克隆技术,将两段X基因扩增片段一步克隆入经Hind III和Xba I双酶切的pcDNA3.0真核表达载体,以构建重组真核表达载体pcDNA-HBX;采用Hind III和Xba I双酶切和DNA序列测序筛选鉴定重组载体;pcDNAHBx转染HepG2细胞,Western blot检测pcDNA-HBx转染细胞的HBx蛋白质表达。结果 :InFusion PCR引物分两段扩增获得全长HBx基因序列;In-Fusion获得克隆的pcDNA-HBx经双酶切筛选得到阳性重组质粒,测序分析证实插入序列正确;转染pcDNA-HBX的HepG2细胞,Western blot证实表达HBx蛋白质。结论 :以血清HBV DNA为模板克隆HBV X基因,构建了表达HBV HBx蛋白质的真核表达载体,为HBx蛋白质的生物学功能研究提供支持。     

山西省晋北地区蔬菜灰霉病菌对乙霉威的抗药性检测

通过对山西省晋北地区蔬菜主要种植区灰霉病菌对乙霉威的抗药性检测,结果表明:32个灰霉病菌菌株时乙霉威的总体抗性频率为68.75%,其中有10株敏感菌株、4株低抗菌株、1株中抗菌株、10株高抗菌株、7株特高抗菌株.     

小鼠骨桥蛋白重组腺病毒的体外构建及表达

目的:构建表达小鼠骨桥蛋白(osteopontin,OPN)基因的重组腺病毒载体,并在主动脉平滑肌细胞(smooth musclecells,SMC)中初步探索其参与血管重塑的相关机制。方法:提取FVB/N遗传背景小鼠的主动脉总RNA,利用针对OPN基因序列设计的特异性引物,应用聚合酶链式反应(polymerase chain reaction,PCR)扩增OPN基因,将PCR产物酶切后连接入腺病毒穿梭质粒pShuttle-CMV载体中,构建pShuttle-CMV-OPN重组穿梭质粒。PCR及测序鉴定正确后,经酶切线性化转染携带腺病毒骨架载体pAdEasy-1的BJ5183感受态细胞进行同源重组,获得pAd-CMV-OPN重组腺病毒质粒。利用磷酸钙法将线性化的pAd-CMV-OPN质粒转染人源胚肾293AA (human embryo kidney 293AA,HEK293AA)细胞进行包装和生产,获得携带OPN基因的重组腺病毒。通过PCR检测病毒原液中重组OPN质粒的包装情况。利用已构建的OPN重组腺病毒感染小鼠SMC,初步探究OPN对血管重塑相关蛋白的调控。结果:成功构建携带OPN基因的重组腺病毒质粒,经磷酸钙-DNA共沉淀转染HEK293AA细胞后,7天可见明显的细胞病变效应,8天后约有60%细胞脱落,收集细胞,反复冻融后分装冻存。与空载体病毒相比,过表达OPN显著上调血管重塑相关蛋白I型胶原(collagen I,Col I)和基质金属蛋白酶2(matrix metalloproteinases 2,MMP-2)的表达。结论:利用pAdEasy-1腺病毒载体系统成功构建携带OPN基因的重组腺病毒,在SMC中OPN可能通过上调Col I与MMP-2参与血管重塑。     

耐甲氧西林金葡菌检测方法比较

目的:研究比较不同方法检测(MRSA)的效果。方法 :将从临床感染标本分离获得的148株金葡菌菌株分别采用头孢西丁、苯唑西林纸片扩散法、基因聚合酶链反应(PCR)法检测MRSA菌株,以采用基因聚合酶链反应(PCR)法作为判断MRSA的"金"标准。并用琼脂稀释法测定148株葡萄球菌对青霉素、红霉素等10多种抗菌药物的药物敏感性。结果 :头孢西丁纸片法对MRSA的检测灵敏度为93.18%,特异度为95%,漏诊率为6.82%,误诊率为5%,Youden指数为0.818;苯唑西林纸片法对MRSA的检测灵敏度为81.82%,特异度为76.67%,漏诊率为18.18%,误诊率为23.33%,Youden指数为0.585,说明苯唑西林纸片法对MRSA的检测准确性低于头孢西丁纸片法。结论:PCR技术检测MRSA快速且敏感性和特异性强,可作为首选鉴别方法。头孢西丁纸片扩散法使用方法简单,对实验条件无特殊要求,较苯唑西林纸片扩散法结果更准确。     

MALDI-TOF MS与16SrDNA方法对肠杆菌科微生物鉴定比较分析

为了研究基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法鉴定肠杆菌科微生物及其对微生物系统分类学相关分析的准确性,采用MALDI-TOF MS对金桔表面分离的10种肠杆菌科微生物进行蛋白质图谱收集,通过比对图谱特征峰实现微生物的鉴定与系统进化学分析;同时对10种微生物进行16SrDNA提取与测序,得到分子生物化学水平鉴定,并采用邻近连接法对16SrDNA序列做系统进化树分析。结果表明:MALDI-TOF MS与16SrDNA测序对10种肠杆菌科微生物鉴定结果中,克雷伯菌属2株菌鉴定种级有偏差,其他8种一致;MALDI-TOF MS与16SrDNA测得的数据都可计算微生物相关性,从而得到微生物系统发育树,两株系统发育树中同属级微生物归类的亲缘位置与方向一致,但不同属肠杆菌科微生物的亲缘距离与位置差异较大。MALDI-TOF MS法鉴定农产品表面微生物具有快速、准确的特点,但准确建立系统发育相关性还需要扩大数据库和优化算法。     

湘江江岸土样中放线菌分离菌株抗菌活性研究

本文采用分散和差速分离法从湘江江岸的土样中分离得到36株放线菌,使用琼脂移块法测定了它们对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、黑根霉和黑曲霉的拮抗作用.结果表明,共有17个菌株对这些指示菌有拮抗作用,并且对金黄色葡萄球菌有抑制作用的菌株基本上都对枯草芽孢杆菌有抑制作用,对枯草芽孢杆菌有抑制作用的菌株基本上都对金黄色葡萄球菌也有抑制作用,然后通过研究菌落形态、菌丝和孢子丝观察、细胞壁的化学组分分析和16s rRNA基因序列对抗菌谱较广的3-2菌株进行了鉴定,初步鉴定为Streptomyces fradiae的一个菌株.     

LPS刺激对人结肠癌SW480细胞基因表达的影响

目的研究结直肠癌细胞炎症模型中基因表达的变化以及所涉及的信号通路。方法利用脂多糖(LPS)刺激SW480细胞1、3、6小时后提取RNA构建测序文库进行转录组测序,对表达有变化的基因归类,并进行京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析,对分析结果进行实时荧光定量PCR验证。结果发现LPS刺激SW480细胞1、3、6小时后,3个时间点表达均有显著变化的基因有250个。对这些差异表达的基因进行了KEGG信号通路富集分析发现利用脂多糖刺激构建的炎症模型里,TNF-α激活和NF-κB信号通路活化最显著。利用癌症基因组图谱(TCGA)数据库进一步对表达持续上调或下调的基因进行分析发现,部分基因在结肠癌标本中的表达趋势与LPS刺激结肠癌细胞引起的表达变化趋势一致。结论在利用脂多糖刺激构建的结肠癌细胞炎症模型中,炎症过程中的基因表达变化可能与结肠癌的发生发展密切相关。     

Y-△降压起动电路的改进设计

Y-△降压起动在大功率的笼型异步电动机起动控制广泛应用。常用的Y-△降压起动电路经常发生接触器主触点烧毛、熔焊的故障,影响电路工作的可靠性。本文在对常用Y-△降压起动电路进行分析的基础上,采用PLC进行了改进设计。     

Effect of glutaraldehyde fixation on bacterial cells observed by atomic force microscopy

Atomic force microscopy (AFM) is a promising microscopy technique that can provide high-resolution images of bacterial cells without fixation. Three species of bacteria, Xanthomonas campestris, Pseudomonas syringae, and Bacillus subtilis, were used in this study. AFM images were obtained from unfixed and glutaraldehyde-fixed cells, and cell height was measured. The mean height of bacterial cells prepared by fixation was higher than that of those prepared by nonfixation. However, the height changes were different between Gram-negative and Gram-positive bacteria: the mean height of two fixed Gram-negative bacteria, X. campestris and P. syringae, increased by 112.31 and 84.08%, respectively, whereas Gram-positive bacterium, B. subtilis, increased only by 38.79%. The results above indicated that glutaraldehyde fixation could affect the measured height of cells imaged by AFM; further more, the effect of glutaraldehyde fixation on the measured height of Gram-negative bacterial cells imaged by AFM seemed much more than on that of Gram-positive bacterial cells.     

Biodeterioration potentials of microorganisms isolated from car engine lubricating oil

Biodeterioration potentials of bacteria isolated from used car engine lubricating oil were examined using both used and unused oils as substrate. Used oil served as a better substrate for growth of the bacteria than unused oil. The bacterial isolates were identified as Bacillus, Corynebacterium, Actinomyces, Micrococcus, Serratia, Citrobacter, Edwardsiella, Pseudomonas, Nocardia and Acinetobacter species. Fungal genera isolated from the oil were Cladosporium, Aspergillus, Cephalosporium, Mucor, Monosporium, Penicillium and the yeast Saccharomyces. Incubation of the bacteria at different temperatures for 48 hours showed that 100% grew at 30°C, 56% grew at 45°C, 44% grew at 55°C, 31% grew at 60°C while 25% grew at 70°C and survived at 80°C. These results indicate that lubricating oil in service is more prone to biodeterioration than unused oil.     

纳米碳化钛颗粒对纳米氧化锆增韧氧化铝基陶瓷刀具材料的影响

研究了纳米TiC对陶瓷刀具材料Al203 - 3Y - Zr02 - TiCn的微观结构和力学性能的影响.结果表明,A5Tn20Z在烧结温度为1650℃、烧结压力为30MPa和保温时间为30min时的力学性能最好,抗弯强度904.7MPa,断裂韧度4.16MPa· m1/2,维氏硬度17.43GPa.     

Proteome analysis in the study of the bacterial heat-shock response

In recent years, it has become clear that, in addition to the regulation of the expression of specific genes, there are global regulatory systems that control the simultaneous expression of a large number of genes in response to a variety of environmental stresses. The first of these global control systems, and of substantial importance, is the heat-shock response. The heat-shock response is characterized by the induction of a large set of proteins (heat-shock proteins-HSPs) upon shifts to higher temperature and upon exposure to conditions in which proteins are denatured (i.e., alcohols, heavy metals). The heat-shock response is universal and many of the heat-shock proteins are highly conserved among species. In bacteria, the heat-shock response has been studied extensively in several Gram-positive bacteria (Bacillus subtilis) and in the Gram-negative bacteria (i.e., Escherichia coli, Agrobacterium tumefaciens). The first recognition of the molecular abundance of the bacterial heat-shock proteins took place with the introduction of high-resolution two-dimensional polyacrylamide gels (2D gels) to analyze complex mixtures of cellular proteins. Two-dimensional gels, followed by mass spectrometry, were used to define the heat-shock stimulons in several bacteria, and to study the regulatory elements that control the heat-shock response. Here, we review the heat-shock response and its regulation in bacteria. The review will emphasize the use of proteome analysis in the study of this response, and will point out those open questions that can be investigated with proteomics, including mass spectrometry techniques.     

Improved sectioning and ultrastructure of bacteria and animal cells embedded in lowicryl

Lowicryl K4M-embedded Gram-positive and Gram-negative bacteria have a tendency to separate between the cell surface and the resin. This often leads to distortion of bacteria and more especially of mycobacteria. We describe attempts made to overcome this technical problem. Different assays were made on Bacillus subtilis, Escherichia coli, and Mycobacterium avium: (1) Modification of the bacterial surface by coating of bacteria with proteinic compounds; (2) treatment of bacteria with metallic salts known to modify cell wall polysaccharides; and (3) comparison between Lowicryl K4M and HM20. Conditions have been found in which the separation of all bacterial species from the resin is abolished. The most important factor appeared to be the treatment of bacteria before dehydration, with 0.5% uranyl acetate for 30 min. The second most important factor, especially for M. avium and to a lower extent for Gram-negative bacteria, was the use of Lowicryl HM20. Pre-embedding in gelatin instead of agar improved sectioning of M. avium, but had no effects on the other bacterial species. These conditions applied to macrophages infected with Shigella dysenteriae or M. avium also gave excellent results. In addition to sectioning improvement of bacteria, uranyl acetate improved the ultrastructure of bacteria and macrophages. All organelles were more clearly delineated and, hence, more easily identified. Finally, it was shown that UA treatment did not affect immunogold labeling of a variety of antigens.     

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION : The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.     

Chemical and thermal denaturation of crystalline bacterial S-layer proteins: an atomic force microscopy study

Crystalline monomolecular cell surface layers, S-layers, are one of the most common outermost cell envelope components of the prokaryotic organisms (bacteria and archaeda) that protects them from competitive habitats. Since isolated S-protein subunits are able to re-assemble into crystalline arrays on lipid films and solid supports making biomimetic surfaces, S-layer technology is currently used in nanobiotechnology. An important aspect of the biomimetic surfaces built with S-layers is their stability under extreme solvent conditions or temperature. Chemical (pH, alcohol) and physical (thermal) denaturant conditions were employed to test the stability of S-layers. Recrystallized bacterial surface layers from Bacillus sphaericus (SbpA) on hydrophilic silicon wafers loses the crystalline structure at 80% ethanol/water mixtures, the change in structure being reversible after treating the surface with buffer solution. SbpA on silicon supports denatures at pH 3 and at 70 degrees C, and the process is irreversible. Cross-linking of SbpA enhances the stability for high ethanol and acidic conditions, but it does not improve thermal stability. Recrystallized SbpA on secondary cell wall polymer (SCWP), a natural environment for the protein layer, is more resistant to ethanol and pH exposure than recrystallized SbpA on hydrophilic silicon supports. Atomic force microscopy (AFM) was used to monitor the loss of stability and the changes in protein layer conformation.     

The influence of probiotics of different microbiological composition on histology of the gastrointestinal tract of juvenile Oncorhynchus mykiss

This article presents results of the influence of three probiotic feed additive of various microbiological composition: Bacillus subtilis (VKPM B-2335); B. subtilis (OZ-2 VKPM-11966) + Bacillus amyloliquefaciens (OZ-3 VKPM-11967); Lactobacillus acidophilus (VKPM B-3235) on the growth and histology of the organs of the gastrointestinal tract of juvenile Oncorhynchus mykiss by morphometric parameters. These probiotic bacteria are the most commonly used in aquaculture. The effect of the probiotic feed additive led to the increase in fish growth and influenced different sections of the gastrointestinal tract. The biggest change was found in the mid intestine and the reliable difference compared with the control diet was obtained at the following parameters: lamina propria width, intraepithelial lymphocytes number of prismatic epithelium and goblet cells area. The changes in the pyloric appendages were less obvious but reported as playing an important functional role in digestion. The liver preserved normal functional structure in all series of the experiment except for the group with L. acidophilus, where hepatocyte small-drop vacuolization was observed. That might be connected with the change of the digest activity resulting from a decrease in secretory activity of the intestinal exocrinocytes. The use of all probiotic feed additives led to a similar change in morphometric parameters in all groups, which suggests a decrease in the immune response.