金黄色葡萄球菌特异性PCR检测靶点的自动化筛选

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。     

水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。     

查尔酮合酶基因的克隆、全序列分析及在大肠杆菌中的高效表达

类黄酮是植物中的一种重要的次级代谢产物,它与植物的花色形成有关。查尔酮合酶(Chalcone synthase,CHS)是类黄酮合成途径中的一个关键酶,在植物体内,CHS表达量的增加或减少都可能改变花的颜色。从矮牵牛(Petunia hybrida)花瓣的cDNA中克隆到了CHS—A基因,进行了全序列分析,并与国外已报道的CHS—A序列进行了同源性比较。结果表明,克隆的CHS-A基因长为1170bp,编码一个由389个氨基酸组成的多肽,与国外已报道的CHS—A同源率高达99％。此外,还在大肠杆菌中实现了CHS—A基因的高效表达。CHS—A基因的成功克隆与表达为研究CHS—A基因对植物花色的影响打下了一个良好的基础。     

非洲菊查尔酮合酶基因的克隆、序列分析及在大肠杆菌中的表达

利用RT-PCR方法,从非洲菊(Gerbera hybrida)花瓣的CDNA中克隆到了查尔酮合酶(Chalcone Synthase,CHS)基因CHS,进行了序列分析。结果表明,克隆到的CHS基因全长为1197bps,编码一个由398个氨基酸残基组成的多肽,与Helariutta等发表的非洲菊查尔酮合酶CHSI基因的CDNA序列的CHS基因同源性高达99％。进一步将该基因克隆到表达载体pET32a上,经IPTG诱导表达,得到高效表达的融合蛋白。     

生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。     

荧光假单胞菌天冬氨酸转氨酶的基因克隆及其在大肠杆菌中的表达

采用克隆基因测序技术,从荧光假单胞菌GcM5-1A基因组文库中筛选到了天冬氨酸转氨酶的编码基因aspC。通过聚合酶链式反应（PCR）扩增目的基因,插入pET-15b构建重组表达质粒pET-15bAAT,转化E.coli BL21(DE3),IPTG诱导天冬氨酸转氨酶在大肠杆菌中高效表达,利用亲和层析法初步分离纯化了重组蛋白。生物活性分析表明,纯化的重组天门冬氨酸转氨酶具有氨基转移活性。     

苦荞查尔酮合成酶基因CHS的结构及花期不同组织表达量分析

采用同源克隆、染色体步移和RT-PCR技术,首次克隆到苦荞查尔酮合酶基因（CHS）的全长DNA序列和cDNA开放阅读框（ORF)序列. 序列分析表明,苦荞CHS DNA序列（GU172165）全长1 632 bp,含1个445 bp的内含子;cDNA编码区（HM852753）全长1 188 bp,编码395个氨基酸,命名为FtCHS. 生物信息学分析表明,FtCHS和推导的氨基酸序列与其它植物CHS基因同源率在95%以上,含有CHS多基因家族的标签序列（GFGPG）、活性位点、底物结合口袋位点和环化反应口袋位点. 半定量RT-PCR分析苦荞花期FtCHS空间表达模型表明,其表达量未成熟种子＞叶＞茎＞花＞根＞成熟种子,与苦荞芦丁含量的分布基本一致,具有组织特异性.     

水蕨(Ceratopteris thalictroides)查尔酮合成酶基因的克隆和表达（英文）

查尔酮合酶(chalcone synthase,CHS)是植物类黄酮化合物合成的关键酶,有关蕨类植物CHS基因的序列及功能信息尚不完善。本研究采用快速扩增c DNA末端(RACE)技术克隆获得了模式蕨类植物——水蕨(Ceratopteris thalictroides)Ct CHS基因(Gen Bank登录号:JX027616.1),其c DNA序列全长为1616 bp,具有3个外显子和2个内含子,开放阅读框(ORF)为1215 bp,编码404个氨基酸。进化树分析表明,Ct CHS与问荆(Equisetum arvense)、松叶蕨(Psilotum nudum)和3种薄囊蕨的查尔酮合成酶基因聚为一枝,说明这些蕨类植物亲缘关系较近且为单系起源。通过构建原核表达体系成功获得Ct CHS蛋白的多克隆抗体并用于免疫印迹分析,结果表明Ct CHS基因的表达明显受紫外光(UV)诱导。Ct CHS基因的克隆与表达分析为进一步研究水蕨类黄酮化合物的合成及其调控机制提供了依据。     

西藏小型猪Leptin及其受体胞外区片段的克隆及原核表达

目的克隆及原核表达西藏小型猪瘦素（Leptin）成熟肽及瘦素受体胞外区片段。方法根据西藏小型猪瘦素序列（GenBank号：GQ240885.1）和猪瘦素受体基因胞外域序列（GenBank号：AF167719.1）分别设计并合成两对引物扩增瘦素、瘦素受体基因胞外域编码区1654-2319位片段,以西藏小型猪组织总RNA为模板,经反转录-聚合酶链反应（RT-PCR）方法获得了特异性片段。再以该两个特异性片段为模板,另外设计两对带有BanHⅠ和HidⅢ酶切位点的套式引物分别扩增瘦素64-504位（成熟肽编码区）和瘦素受体基因胞外域编码区1655-2314位的cDNA片段,将该两片段克隆入pMD18-T载体并转化感受态细菌E.coli DH5α测序并永久保存。此两片段经酶切后克隆到表达载体pRSET A的BamHⅠ和HindⅢ两酶切位点之间,构建重组质粒pR-OB和pR-OBR-a并在大肠杆菌E.coli BL21（DE3）中表达,SDS-PAGE电泳鉴定表达产物。结果在IPTG诱导下促使重组菌pR-OB表达了相对分子质量约18×103左右的融合蛋白;重组菌pR-OBR-a表达了相对分子质量约27×103左右的融合蛋白。结论说明重组质粒pR-OB、pR-OBR-a在大肠杆菌BL21（DE3）中分别可表达西藏小型猪瘦素成熟肽、瘦素受体片段蛋白,为进一步研究瘦素、瘦素受体功能和应用提供了基础。     

决明查尔酮合成酶全长cDNA的克隆及其原核表达载体的构建

从决明子叶中提取查尔酮合成酶(Chalcone synthase)总RNA,经过RT-PCR获得查尔酮合成酶cDNA,纯化后与pMD18-T载体连接,转化大肠杆菌Top10,获得了查尔酮合成酶全长基因序列.序列测定表明,查尔酮合成酶全长核苷酸长度为1 173 bp,编码390个氨基酸.与GenBank中已发表序列EU430077进行比较,核苷酸同源性为100%.将该基因片段克隆到原核表达栽体pET-28a(+)中,构建重组质粒pET-28a(+)/Chalcone synthase,所获重组质粒经过双酶切、PCR和序列测定,证实含有目的片段,且连接、构建正确,为查尔酮合成酶的进一步表达奠定了基础.     

Nonenzymatic modifications of amino sugars in vitro

Browning reactions of amino sugars were observed in a variety of sterile pH buffers at 25-37 degrees C. These reactions were signaled by an increase in absorbance at 273 nm, followed by an increase in absorbance at 320-360 nm. The reactions were maximal at pH 7.0 in phosphate buffer. Acidic solutions (pH less than 2.2) of 50 mM D-glucosamine hydrochloride gave only a negligible reaction and 2-acetamido-2-deoxy-D-glucose was unreactive. Half of the D-glucosamine in a 100 mM solution in sterile 0.2 M sodium phosphate buffer, pH 7.4, at 37 degrees C decomposed or was transformed in 27 h. A comparison of reactivity in generating A273 and A340 chromophores showed D-mannosamine greater than D-galactosamine greater than D-glucosamine. Permanganate oxidation of incubated glucosamine solutions afforded a compound which chromatographed like 2,5-pyrazinedicarboxylic acid and gave the same ultraviolet absorption spectrum. This, together with fractionating and thin-layer chromatography of the products of glucosamine incubation, suggests that 2,5-bis(tetrahydroxybutyl)pyrazine is formed as one of the products of autocondensation of D-glucosamine in accord with the report of Candiano et al. (1988, Carbohydr. Res. 184, 67-75) on products formed in glucosamine-lysine incubation mixtures. Formation of products absorbing at 325-360 nm was inhibited by the chelator diethylene-triaminepentaacetic acid. This suggests that the later reactions may be mediated by a metal-stimulated free radical mechanism. After 4 days incubation high molecular weight products with absorbance maxima at 273 nm and 325-360 nm were detected. Some of these were retained by dialysis membranes of molecular weight cut-off greater than 3500 and greater than 12,000.     

Water-soluble chlorophyll protein of Brassica oleracea var. Botrys (cauliflower)

A water-soluble chlorophyll protein was prepared from Brassica oleracea var. Botrys (cauliflower) and purified by (NH₄)₂SO₄ fractionation and by chromatography on a DEAE-cellulose column. The chlorophyll protein contained chlorophylls a and b in the ratio 6:1, and no carotenoids. The molecular weight, determined by means of gel filtration on Sephadex G-100, was 78000. The chlorophyll protein showed absorption peaks at 273, 340, 384, 420, 438, 465, 628, 674 and 700 nm. Since the three bands at 384, 420 and 438 nm all have approximately the same height, the spectrum is different from that of chlorophyll a in organic solvents. The fluorescence of the chlorophyll protein showed a peak at 683 nm, with shoulders at 706 and 745 nm at room temperature, and peaks at 685, 706 and 744 nm at the temperature of liquid N₂. An apo-protein was prepared by removing the chlorophylls with 2-butanone and purified by precipitation with (NH₄)₂SO₄. The apo-protein thus prepared had an absorption band at 273 nm but none at longer wavelengths. The apo-protein could be combined with chlorophylls, forming a chlorophyll protein which had spectral characteristics similar to those of the original.     

The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.     

Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization

We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.     

毛脉酸模不同生长发育期HPLC色谱指纹图谱研究

采用HPLC-DAD方法,梯度洗脱,对10批不同生长发育期毛脉酸模根样品进行主成分分析。色谱条件为:Planetsil C18分析柱(5μm, 200 mm×4.6 mm),预柱Phenomenex ODS-C18(4×3.0 mm ID),柱温40℃,流动相A为甲醇;流动相B为水(磷酸调pH值为2.0),流动相A从30%甲醇到100%甲醇,时间为0~50 min,检测波长254 nm。10批毛脉酸模样品得到的色谱指纹图谱有27个共有峰,其特征峰指纹图谱可分为Ⅰ, Ⅱ, Ⅲ三组:第Ⅰ组包括0~17 min(1~14号峰),第Ⅱ组包括17~35 min(15~24号峰),第Ⅲ组包括35~50 min(25~27号峰)。HPLC-DAD方法分析毛脉酸模主成分,方法准确可靠,其本身具有多成分同时定性的优势;27个共有峰的出峰先后顺序及相对含量极具特征性、专属性,重复性好,形成了毛脉酸模特有的HPLC色谱指纹图谱,可作为毛脉酸模内在质量评价、鉴定及其最佳采收期确定提供科学依据。     

类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑的酶促合成及表征

肌肽(β-Ala-L-His)是一种高效抗氧化剂,广泛应用于生物、化工、医药等领域。应用微水相酶促合成类肌肽,效率高价格低,且具有相似性质,开发前景广阔。本研究以L-丙氨酸和4,5-二羧酸咪唑制备类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑,正交实验下的最佳合成条件为:四氢呋喃:pH8磷酸缓冲溶液=10:1.6(V/V),L-丙氨酸:4,5-二羧酸咪唑=1:3(m/m),α-胰凝乳蛋白酶:底物=1:200(m/m),35oC下磁力搅拌1.5h。硅胶G60薄层色谱(TLC)分离反应产物,Rf=0.81处出现新斑点;刮下该点纯化后进行紫外扫描,高效液相色谱(HPLC)和核磁共振,紫外光谱253nm处吸收明显增强,310nm处出现新吸收峰;253nm、310nm、330nm高效液相色谱保留时间均为4.0min;13C核磁共振显示8组碳原子。结合胰凝乳蛋白酶的催化机理,得出产物结构为4(5)-丙氨酰胺-5(4)-羧酸咪唑。     

高效液相色谱法分析测定越桔果渣中的黄酮甙元

采用高效液相色谱法对越桔果渣中黄酮甙元进行定性和定量分析.用甲醇和水（60：40）以0.8 mL/min流速进行洗脱,368 nm波长检测越桔果渣水解液中黄酮类成分并测定其含量.结果：越桔果渣中黄酮甙元主要有杨梅素和槲皮素,其含量分别为：0.0206%和0.0686%.该方法测定样品简单易行,分离效果佳,灵敏度高,重现性好,结果准确可靠.     

Studies on the toxin of Aspergillus fumigatus. VII. Purification and some properities of hemolytic toxin (asp-hemolysin) from culture filtrates and mycelia.

A hemolytic toxin has been obtained from mycelia and culture filtrates of Aspergillus fumigatus by the procedures that included precipitation with ammonium sulfate, chromatography of DEAE-Sephadex, affinity chromatography on Concanavalin A-Sepharose and gell filtration on Sephadex G-50, G-100 AND G-150. The purified homolytic toxin was homogeneous on immunological and disk electrophoretic analysis, and the toxin from culture filtrates was identical with that from mycelia by the immunodiffusion technique. The hemolytic toxin was obtained for the first time from fungi and designated as Asp-hemolysin. The molecular weight of Asp-hemolysin was estimated to be appoximately 30,000 by the gel-filtration technique and its isoelectric point was found to be around pH 4.0. This Asp-hemolysin contained large amounts of protein and very small amounts of carbohydrate. The UV absorption spectrum of Asp-hemolysin showed a maximum absorption at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm, E 1% 1CM, was 12.4 and the ratio of absorbance at 280 nm to that at 260 nm was 2.3. The optimum pH for the hemolytic activity of the toxin toward chicken erythrocytes was 5.0 at room temperature and it was active in the pH range of 3.5 to 10.5. The optimum temperature was 21 C and about 50% of the activity was lost by incubation at 50 C for 5 min or 45 C for 23 min. The hemolytic activity was remarkably inhibited by Hg2+, Cu2+, Fe2+, Ag1+, iodine and p-CMB, but enhanced slightly by Zn2+ and Co2+.     

竹红菌甲素敏化嘌呤和嘧啶碱的光氧化作用及其影响因素

本工作利用光吸收和高效液相色谱(HPLC)技术研究了甲素对DNA分子中四种碱基A、G、C和T光氧化的敏化作用,发现在反应体系的pH为9.0、甲素浓度为3×10~(-5)mol/L、光照40分钟时,G和T紫外吸收明显降低;HPLC分析发现甲素敏化的G光氧化体系比对照体系多出现一组分峰(滞留时间0.927分钟),该峰用475nm波长检测比260nm波长检测灵敏。根据反应机制推测是G环破裂产物。在反应条件固定时,甲素敏化G的光氧化作用受pH、光照时间及甲素浓度影响极大。单线态氧淬灭剂——叠氮钠浓度在40—110mmol/L可部分抑制甲素敏化G的光氧化作用,>110mmol/L时反应完全被阻断,提示甲素对G光氧化的敏化作用主要通过单线态氧(~1O_2)即Ⅱ型机制起作用。本文还讨论了G光氧化的可能途径。     

Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.