预防新生仔猪大肠菌性腹泻的双价工程菌苗

本文应用遗传工程技术,将猪毒素源性大肠杆菌(ETEC)的粘附素K88与无毒肠毒索LTA~-B~+基因组建成能同时表达两种抗原成分的菌苗;进一步去除抗药性基因,以β-半乳糖苷酶活性作为筛选标记,得到表达水平相似的双价菌苗。菌苗无毒安全,能有效地激发机体的免疫应答,经过人工攻毒试验与田间试验,首次报道了K88与LT协同免疫对大肠菌性腹泻的显著保护力,以及无抗药性基因菌苗在实际中的应用。     

毛细管电泳-单链构象多态性分析检测K-ras基因突变

K ras癌基因的点突变在结直肠癌的发生起重要作用。以异丙醇为聚合反应链转移剂,水相法合成 特性粘度为0.70×10-3m3/kg,分子量为6.5×104的低粘度短链线性聚丙烯酰胺。以6%线性聚丙烯酰胺为 筛分介质,分离温度27℃,分离电压9kV为电泳条件,建立了检测K ras基因突变的毛细管电泳 单链构象多 态性方法。利用该方法检测36例结直肠癌患者肿瘤组织,发现12例K ras基因突变。结果表明:该方法具有 快速、高灵敏的优点,为大规模进行结直肠肿瘤的早期诊断提供了可靠方法。     

毛细管电泳法同时测定复方苦参注射液中三种生物碱含量

应用毛细管电泳法测定了复方苦参注射液中苦参碱、氧化苦参碱、槐定碱的含量。毛细管电泳条件为 :未涂层石英毛细管 ( 5 7cm× 5 0 μm i.d.) ,分离电压为1 2 .5 k V,0 .2 mol/ L Tris- 40 mmol/ L磷酸二氢钠 ( p H5 .5 0 ) - 2 0 %异丙醇为缓冲液 ,检测波长 2 0 0 nm,氢溴酸东莨菪碱为内标。结果表明 ,该法简便、快速、准确 ,重现性好。     

响应曲面优化-毛细管电泳-激光诱导荧光快速检测粪便中诺如病毒

建立了粪便中诺如病毒逆转录PCR-毛细管电泳-激光诱导荧光快速检测方法。根据诺如病毒核酸保守序列选择引物,扩增出特异的PCR产物;采用响应曲面法进行毛细管电泳条件优化,以含有DNA荧光染料SYBR Gold的0.5%甲基纤维素为筛分介质,通过激光诱导荧光法检测诺如病毒的PCR产物。在优化的毛细管电泳条件下,9 min内可完成诺如病毒PCR产物的检测。扩增产物测序后,与基因库中诺如病毒的序列进行同源性比对,一致性达99%。迁移时间的日内和日间相对标准偏差分别为1.1%~1.3%和1.8%~2.6%,已用于粪便中诺如病毒的快速检测。     

基于生物芯片的核酸凝胶电泳仪

为克服传统平板凝胶电泳法操作步骤繁琐,实验时间长,以及采用紫外光源可能对人体造成伤害等缺陷,该文研制了一种基于电泳芯片的快速凝胶电泳仪,仅需DNA点样于电泳芯片后,将电泳芯片置于该电泳仪便可实现DNA样品快速分离及实时在线成像检测。以20、50、100 bp DNA ladder为检测对象,在仪器内对其分离效果进行验证及优化,其最佳电泳条件为电压100 V/cm,琼脂糖质量分数分别为2. 5%、1. 0%、1. 4%。结果表明在14 min内可以实现3种DNA样品的高效分离,DNA迁移距离与分子量的相关系数均高于0. 9,且该仪器具有较高的稳定性。     

牡丹皮中有效成分丹皮酚的毛细管电泳快速检测新方法

采用毛细管电泳高频电导法对丹皮酚进行了快速分离检测。对电泳介质的种类及浓度、操作电压和进样时间等影响因素进行了优化。最佳条件为：分离介质1．0mmol／LH3BO3-3．0mmol／L三乙胺-10％CH,OH（pH=8．0）,分离电压20．0kV,25．0cm位差虹吸进样8．0s。在该条件下。可在4min内实现对丹皮酚的分离检测。线性范围为2．0～105μg／mL,检出限为0．3μg／mL。成功测定了中药牡丹皮中的丹皮酚,回收率达94％～99％。方法简便、快速、灵敏,可用于药物分析。     

毛细管电泳技术快速检测牙周病原菌

建立了毛细管电泳快速检测牙龈卟啉单胞菌(P.g)、齿垢密螺旋体(T.d)、福赛斯坦纳菌(T.f)3种牙周病病原菌种的方法。紫外分光光度法表明,采用无菌吸潮纸尖及PBS缓冲液可以实现牙周病原菌的快速有效提取。对提取的牙周病病原菌种做多聚酶链式反应(PCR)与多重PCR反应,最后以20 cm长,75μm内径的石英毛细管作为分离通道,分离电压4000 V,1.2%羟乙基纤维素(HEC,250 K)为筛分介质,牙周病病原菌菌种P.g,T.d,T.f PCR及多重PCR产物12 min内得到较好分离。结果表明,毛细管电泳与PCR技术相结合,可以实现牙周病原菌种快速鉴定,且检测限低至4.80×10-11ng/μL。方法已用于牙周病原菌菌种的快速检测。     

区带毛细管电泳分离测定大黄提取液中游离蒽醌化合物

采用区带毛细管电泳法分离和同时测定大黄提取液中五种蒽醌化合物的含量。毛细管电泳的分离条件为:50 cm×75μm i.d.未涂层石英分光毛细管,分离电压20kV,0.05 mol/L KH2PO4-Na2HPO4(pH=8.2)缓冲溶液,紫外检测波长为254 nm,以氨基苯磺酸为内标。该法简便,快速,分辨率高,重现性好。     

高效毛细管区带电泳法分离人血清蛋白质的研究

 研究了高效毛细管区带电泳分离人血清蛋白质的电泳行为及实验条件 ,建立了分离血清蛋白质的高效毛细管区带电泳法。血清样品经硼酸缓冲液 (5 0 mmol/L,p H8.80 )稀释后 ,以 0 .1 mol/L硼酸缓冲液 (p H9.3 5 ,含4g/L PEG80 0 0 )为电泳介质 ,在 5 0 μm i.d.× 3 7cm弹性石英毛细管柱 (有效柱长为 3 2 cm)中进行电泳分离 ,以2 0 0 nm紫外波长检测。方法简便、快速、重复性好 ,1 2 min内便可完成对血清中蛋白质的电泳分离。     

高效毛细管电泳电导法快速检测复方维生素B片中的VBl、VBl2、VB6和VC

采用高效毛细管电泳电导法同时分离、测定了复方维生素B片中的主要成分VB1，VB12，VB6和VC的含量。研究了运行缓冲溶液的酸度和浓度、电泳电压、进样时间等因素对电泳的影响。在优化的实验条件下：40mmol/L Tris-4mmol/L H3BO3(pH8．0)的缓冲溶液中加入0．30mmol/L CTAB(溴化十六烷基三甲基铵)，分离电压为15kV，上述4组分在5min内得到良好的分离。维生素B1，B12，B6和VC的线性范围分别为5．5～1．0mg/mL；15～1．5mg／mL;1．0～0．40mg／mL和6．6～0．80mg／mL；检测限分别为0．80μg/mL，4．0μg/mL，0．50μg/mL，2．9μg/mL；5次测定峰高的相对标准偏差分别为2．2％，1．6％，3．9％，2．8％。5次测定的平均回收率分别为99％，94％，l00％，97％。     

Capture efficiency of Escherichia coli in fimbriae-mediated immunoimmobilization

Capturing pathogens on a sensor surface is one of the most important steps in the design of a biosensor. The efficiency of a biosensor at capturing pathogens has direct bearing on its sensitivity. In this work we investigated the capturing of Escherichia coli on substrates modified with antibodies targeting different types of fimbriae: K88ab (F4), K88ac (F4), K99 (F5), 987P (F6), F41, and CFA/I. The results suggest that all these fimbriae can be used for the efficient immobilization of living E. coli cells. The immobilization efficiency was affected by the purity and clone type of the antibody and the fimbriae expression level of the bacteria. For a specific fimbriae type, a higher immobilization efficiency was often observed with the monoclonal antibodies. Immunoimmobilization was utilized in an antibody microarray immersed in a mixed culture of pathogens to demonstrate the rapid and simultaneous label-free detection of multiple pathogens within less than 1 h using a single test. The capture rate of living pathogens exceeds a single bacterium per 100 × 100 μm(2) area per 0.5 h of incubation for a bulk concentration of 10(5) cfu/mL.     

Recombinant bivalent live vaccines against neonatal colibacillosis in piglets

The genes of colonization factor K88 and avirulent heat-labile enterotoxin LT A-B+ of enterotoxigenic E. coli (ETEC) have been ligated, and a vaccine strain containing recombinant plasmid that efficiently expresses two antigens has been obtained. Using the activity of beta-galactosidase as a selection marker instead of drug resistance, another bivalent vaccine strain with the same expression level has also been constructed. The vaccine strains have no toxicity and do not cause any adverse reactions. In challenge study and field trials, a high degree of protection from colibacillosis was afforded to piglets when their dams were immunized orally or parenterally. Practice of bivalent live vaccines including colonization factor and enterotoxin antigens and without antibiotic resistance gene shows effective.     

Identification of Escherichia coli by detection of hydroquinone and uracil in the urine system

For rapid identification of Escherichia coli, changes of urinary metabolites incubated with E. coli were investigated by gas chromatography--mass spectrometry. Hydroquinone and uracil were detected and the normal urinary constituent 4-deoxythreonic acid was found to diminish in urine incubated with E. coli. Hydroquinone could not be detected in urine incubated with Klebsiella pneumoniae, Serratia marcescens or Pseudomonas aeruginosa. Although uracil was detected in normal urine, urine incubated with E. coli showed an increased uracil level. Urine incubated with K. pneumoniae, S. marcescens or P. aeruginosa evidenced no such change. A decrease of 4-deoxythreonic acid was noted in urine incubated with S. marcescens or P. aeruginosa. In 7.0 X 10(7) cells of E. coli, 0.33-2.36 micrograms of hydroquinone and 13.4-42.0 micrograms of uracil were detected after 3 h of incubation at 38 degrees C, and production was not changed after 4, 5 or 8 h of incubation. These results suggest that the detection of hydroquinone and uracil in urine is useful for rapid identification of E. coli.     

B subunit of cholera toxin produced in Escherichia coli

An engineered E. coli strain containing high expression level of CT-B subunits has been obtained by the application of recombinant DNA techniques. The B subunit can be secreted into the medium and reaches 20-40 micrograms/ml when this strain is incubated in a 50 l fermentation tank. The CT-B subunit purified with affinity chromatography in E. coli has the same characters as the natural CT-B subunit in molecular weight, N terminal amino acid analysis and antigenicity. The CT-B subunit has good immunogenicity and can be used as a preparation for protecting against diarrhea caused by V. cholera and enterotoxigenic E. coli. It can also be used as a vector for hepatins.     

Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens

Escherichia coli O148 is a nonencapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide of this bacterium is considered both a virulence factor and a protective antigen. It is built up of the linear tetrasaccharide repeating unit [3)-α-L-Rhap-(1→2)-α-D-Glcp-(1→3)-α-D-GlcNAcp-(1→3)-α-L-Rhap-(1→] differing from that of the O-SP of Shigella dysenteriae type 1 (SD) only in that the latter contains a D-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SPs of these bacteria indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of mice with these conjugates induced anti-O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the LPSs of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-SPs that was further supported by Western-blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SPs of both bacteria.     

Probing the transmembrane potential of bacterial cells by voltage-sensitive dyes.

Fluorescent dyes have been widely employed as optical indicators of the membrane potential difference in cells, isolated organelles and lipid vesicles that are too small to make microelectrode measurements feasible. We describe here the application of a carbocyanine dye, 3,3'-dipropylthiodicarbocyanine iodide [DiS-C3-(5)], to monitor the transmembrane potential changes induced by a variation of the K+ concentration for the cells of Escherichia (E.) coli and photosynthetic bacterium Rhodospirillum (R.) rubrum. The cells were first incubated in buffers containing DiS-C3-(5) and K+ ions of various concentrations until the fluorescence intensity reached a constant value. Valinomycin was then added to the solution, which caused changes in the fluorescence intensity, depending on the K+ concentrations. The membrane potential is shown to have a linear relationship with the fluorescence intensity of DiS-C3-(5). The results demonstrate that the K+ concentrations inside intact cells are 4.6 mM and 5.3 mM for E. coli and R. rubrum, respectively. The diffusion potentials of K+ ions were determined using the Nernst equation over the range of -1.3 mV to 44 mV, corresponding to K+ concentrations of 5 mM -25 mM outside of the cells.     

Dynamic modification of microorganisms by pyrenebutanoate for fluorometric detection in capillary zone electrophoresis

Pyrenebutanoate, a fluorescent amphiphilic probe, is suggested here as a capillary zone electrophoresis (CZE) buffer additive for dynamic modification and analysis of microbial cells. Mixed cultures of microorganisms Escherichia coli, Candida albicans, Enterococcus faecalis and Staphylococcus epidermidis were concentrated, resolved by CZE and detected. Using UV excitation for on-column fluorometric detection, a detection sensitivity for the microorganisms on the order of from one to tens of injected cells was achieved.     

碱促进β-酮酸酯与2-苯二甲酰亚氨基丙烯酸甲酯的共轭加成反应合成α-氨基酸衍生物

以K_2CO_3为碱,β-酮酸酯与2-苯二甲酰亚氨基丙烯酸甲酯经共轭加成反应合成了一系列α-氨基酸衍生物,收率88%~99%,d/r值1.2∶1~1.3∶1,其结构经1H NMR,13C NMR和HR-MS(ESI-TOF)确证。     

Temperature-dependent coordination in E. coli manganese superoxide dismutase

Two different temperature dependences of the manganese(II) high-field electron paramagnetic resonance spectrum of manganese superoxide dismutase from E. coli were observed. In the 25-200 K range, the zero-field interaction steadily decreased with increasing temperature. This was likely due to the thermal expansion of the protein. From these results, it was possible to deduce an approximately r(-)(2.5) dependence of Mn(II) zero-field interaction on ligand-metal distance. At temperatures above 240 K, a distinct six-line component was detected, the amplitude of which decreased with increasing temperature. On the basis of similarities to the six-line spectrum observed for the azide-complexed E. coli manganese superoxide dismutase, the newly detected six-line spectrum was assigned to a hexacoordinate Mn(II) center resulting from the coordination of a nearby water molecule to the normally five-coordinate center. The changes in enthalpy and entropy characterizing the hexacoordinate-pentacoordinate equilibrium in the 240-268 K range were -5 kcal/mol and -24 cal/mol.K, respectively. The structural implications of the zero-field parameters of the newly found hexacoordinate form in comparison to those of the Mn(II) centers in concanavalin-A and manganese-containing R. spheroides photosynthetic reaction centers and the values predicted by the superposition model are discussed.