水稻中一种抗真菌蛋白的分离与特性分析

从萌发的水稻种子中分离并纯化了一种能抑制多种病原真菌生长的蛋白（简称为ＲＡＦＰＩ），在马铃薯葡糖琼脂培养基上，２０μｇＲＡＦＰＩ可明显抑制木霉（Ｔｒｉｃｈｏｄｅｒｍａｒｅｅｓｅｉ）菌丝的生长，此蛋白还具有较广的抗真菌菌谱，其分子量为１６．５９ＫＤ，等电点为８．７，组成成分中富含Ｐｒｏ、ＧｌＸ和Ａｓｘ，经测序得到了其Ｎ端２０个氨基酸序列。     

水稻抗真菌蛋白是否就是一种胰蛋白酶抑制剂

水稻抗真菌蛋白是否就是一种胰蛋白酶抑制剂王槐春（中国军事医学科学院情报研究所北京１００８５０）《高技术通讯》今年第２期２２—２６页发表了刘虹等人的《水稻中一种抗真菌蛋白的分离与特性分析》一文，报道了从萌发的水稻种子中分离和纯化出一种能抑制多种病原真菌...     

抗真菌蛋白Rs-AFP2基因定点突变及其对大肠杆菌中表达的影响

为了研究遗传密码子对表达调控的影响,利用PCR重叠延伸法,对萝卜抗真菌蛋白Rs-AFP2基因编码序列区的部分核苷酸进行沉默突变,构建突变体Rs-AFPm.序列分析表明,PCR产物全长240bp,有一个阅读框,编码的蛋白由29个氨基酸的信号肽和51个氨基酸的抗真菌蛋白组成.突变体与突变前的Rs-AFP2基因相比,在编码区第3号氨基酸Lys相差一个碱基(TTG→TTA),第5号氨基酸Gln相差一个碱基(CAG→CAA),第6号稀有密码子Arg相差两个碱基(CAG→CGA).重新合成引物,将切除信号肽的Rs-AFP2基因和Rs-AFPm基因与原核表达载体pET-21b(+)分别重组到大肠杆菌BL21菌株.IPTG诱导后,二者均得到了表达.软件分析显示,突变前pETAFPo表达产物占全菌蛋白的3%,突变后pETAFPm的表达产物占全菌蛋白含量的8%;表达蛋白主要以包涵体的形式存在,包涵体经超声波破碎后,蛋白质复性,抑菌结果表明,pETAFPm表达产物的抑菌半径大于pETAFP2表达产物的抑菌半径.这些都说明改造后的Rs-AFPm基因与Rs-AFP2基因相比,已有效地提高表达量.     

抗夏菌蛋白Rs—AFP2基因定点突变及其对大肠杆菌中表达的影响

为了研究遗传密码子对表达调控的影响，利用PCR重叠延伸法，对萝卜抗真菌蛋白Rs-AFP2基因编码序列区的部分核苷酸进行沉默突变，构建突变体Rs-AFPm。序列分析表明，PCR产物全长240bp，有一个阅读框，编码的蛋白由29个氨基酸的信号肽和51个氨基酸的抗真菌蛋白组成。突变体与突变前的Rs-AFP2基因相比，在编码区第3号氨基酸Lys相差一个碱基(TTG→TTA)，第5号氨基酸G1n相差一个碱基(CAG→CAA)，第6号稀有密码子Arg相差两个碱基(CAG→CGA)。重新合成引物，将切除信号肤的Rs-AFP2基因和Rs-AFPm基因与原核表达载体pET-21b( )分别重组到大肠杆菌BL21菌株。IPTG诱导后，二者均得到了表达。软件分析显示，突变前pETAFPm表达产物占全菌蛋白的3％，突变后pETAFPo的表达产物占全菌蛋白含量的8％；表达蛋白主要以包涵体的形式存在，包涵体经超声波破碎后，蛋白质复性，抑菌结果表明，pETAFPm。表达产物的抑菌半径大于pETAFP2表达产物的抑菌半径。这些都说明改造后的Rs-AFPm基因与Rs-AFP2基因相比，已有效地提高表达量。     

在大肠杆菌中表达具有抗黄曲霉活性的鲎素

人共设计并合成了抗真菌多肽鲎素基因。将合成的鲎素基因首先克隆到ｐＵＣ１８载体上,利用通用引物测序,获得了与预期碱基序列完全一致的目的基因。再将鲎素基因转入大肠杆菌,诱导表达融合蛋白。３７℃下诱导表达的ＧＳＴ－鲎素融合蛋白完全不溶解,以包涵体的形式出现;而在３０℃诱导表达时,有少部分融合蛋白是可溶的。     

华根霉(Rhizopus chinensis)前导肽脂肪酶基因的克隆及其在Pichia pastoris中的表达

通过3次PCR程序克隆得到了华根霉(Rhizopus chinensis CCTCCM201021)脂肪酶全基因序列,该基因的开放阅读框长1170bp,不含内含子,编码一个389个氨基酸残基的蛋白质,包括26个氨基酸的信号肽,94个氨基酸的前导序列和269个氨基酸的成熟肽,其推断的氨基酸序列与一些已报道的根霉脂肪酶序列同源性为86%.在巴斯德毕赤酵母(Pichia pastoris)GS115中分泌表达前导肽序列.SDS-PAGE分析表明表达蛋白的分子量约37kD.N-端氨基酸序列分析表明该重组蛋白分泌过程中前导序列N-端的67个氨基酸被切割掉,表达的重组脂肪酶由前导序列C-端的27个氨基酸和成熟肽的269个氨基酸组成.发酵132h后上清中重组脂肪酶的表达量最高,蛋白含量约5.4mg/mL,橄榄油乳化法测水解酶活为161U/mL,其比活比野生型华根霉脂肪酶高约43倍.     

水稻矮缩病毒非结构蛋白Pns11的核酸结合活性分析

水稻矮缩病毒的非结构蛋白Pns11是一个序列非特异性的核酸结合蛋白,其N端有类似锌指蛋白的结构,C端富含碱性氨基酸,为了研究Pns11的核酸结合活性,构建了两个位于可能的锌指结构内的点突变以及四个分别删除部分碱性区氨基酸片段的缺失突变,经测序验证后分别克隆到大肠杆菌表达载体pBV221中,温度诱导表达,用提取包涵体的方法初步纯化突变体蛋白,Western印迹检测这些突变体和Pns11的抗体均有特异反应,Csouthwestern和Northwestern印迹的方法证明六个突变体都保持了核酸结合活性,结果表明N端的类似锌指蛋白的结构不是结合核酸的活 位点,部分缺失C端的碱性氨基酸也不影响其结合。     

中药黄芪蛋白成分的分离和分析

中药黄芪(Huangqi)的茎的抽提液经CM-SFF柱和Sephacryl S-200柱分离纯化,得到一种毒蛋白,用高效凝胶蛋白柱和反相高效液相色谱法分离后,利用光电二极管阵列检测器峰的光谱特性确认分离峰的纯度和蛋白特性,在高效凝胶蛋白柱上制备了少量毒蛋白纯样,测定了毒蛋白分子量和氨基酸组成。     

凤凰木种子毒蛋白的高效液相色谱研究

本文介绍用HPGFC对凤凰木种子中提取的三种毒蛋白分别进行色谱分析。并对分离的蛋白峰进行紫外光谱扫描来确认蛋白的纯度。根据标准分子量曲线,分别得到它们的分子量并与SDS-PAGF所得分子量进行比较,用柱后衍生法测定了三个蛋白各自的氨基酸组成。     

生物蛋白的氨基酸检测分析方法研究

利用氨基酸分析仪,对生物蛋白氨基酸检测方法进行分析研究,确定了准确、有效的检测方法,该检测方法重现性较好,各氨基酸的平均回收率为95%,RSD<3%(n=3)。最小检测量可达30nmol。     

Development of Anticandidal Delivery Systems: (I) Anticandidal Activities of Antifungal Agents and Synergistic Combination with Other Drugs

Susceptibility testing with antifungal agents, e. g., minimum inhibitory concentration (MIC) determination, is performed to obtain reliable data that permit selection of the most suitable agents for treatment of an infective condition. To determine the drugs that provide maximum effectiveness against oral candidiasis, the MICs of various antifungal agents were determined. Also, synergism between two chosen antifungal agents was evaluated, and the effect of benzocaine, an anesthetic, and hydrocortisone, an antiinflammatory agent, on their MICs was examined. It was observed that among all the drugs tested, clotrimazole was the most promising candidate for use as an oral local antifungal. The combination of clotrimazole and chlorhexidine resulted in a decrease in the MIC. While the addition of hydrocortisone to this combination resulted in a slight increase in the MIC, the inclusion of benzocaine resulted in a substantial decrease in the MIC of the antifungal agent combination.     

Oxygen sensor based on the fluorescence quenching of a ruthenium complex immobilized in a biocompatible Poly(Ethylene glycol) hydrogel

An optically based system has been developed for use as an oxygen sensor for a cell culture bioreactor. Electrochemical sensors based on the Clark oxygen electrode are typically used with cell-culture bioreactors. These sensors, however, are subject to long-term drift, due in part to biofouling, and require penetrating the bioreactor with the probe in order to perform a measurement. We report an implantable sensor that, when used with an external fiber-optic probe, takes advantage of the oxygen stimulated fluorescence quenching of dichloro(tris-1,10-phenanthroline) ruthenium (II) hydrate. This fluorophore was immobilized in a photopolymerized hydrogel made from poly(ethylene glycol) diacrylate (PEG-DA), a polymer known to minimize protein and cell adhesion. A low-average molecular weight PEG-DA (MW = 575) was employed to hinder the fluorophore from leaching. The PEG-DA precursor solution contained 40% H/sub 2/O such that, upon polymerization, the gel was already in the hydrated state. Sensor hydrogels stored in H/sub 2/O for several months retained their physical shape and sensitivity to oxygen. The sensor showed a high degree of reproducibility across a range of oxygen concentrations that are typical for cell culture experiments (0-9.1 ppm O/sub 2/), and a linear model produced a strong correlation (R /sup 2/= 0.995) compared with a commercial electrochemical probe. No drift or hysteresis was identified in the sensor across cycles of varying oxygen concentrations in this range.     

Amphotericin B-loaded solid lipid nanoparticles (SLNs) and nanostructured lipid carrier (NLCs): effect of drug loading and biopharmaceutical characterizations

The aim of this study was to further investigate the effect of drug loading, drug entrapment efficiency, the drug release profiles and biopharmaceutical point of views of amphotericin B (AmB) lipid formulations, that is, degree of aggregation by UV-spectroscopy, in vitro hemolytic and antifungal activities. The optimum drug loading was 2.5% by weight corresponded to lipid fraction in formulation. Increasing of the drug entrapment was achieved by blending small amount of phospholipid in solid lipid nanoparticle (SLN) dispersions. All AmB lipid dispersions were less aggregated species and hemolytic response than Fungizone^® indicating that lipid nanoparticles could reduce its toxicity. The sustained release profiles of AmB formulations depended on its aggregated form and entrapment efficiency. Too high AmB loaded (5% w/w) showed a biphasic drug release profile probably due to some amounts of drug deposited on the nanosphere surface including in continuous phase which promptly released. For in vitro antifungal testing, all AmB lipid formulations were equal and more effective than both AmB itself and Fungizone^®. These observations suggested that AmB loaded SLNs, nanostructured lipid carriers and modified SLNs by blending lecithin could enhance AmB solubility, prolong release characteristics, reduce toxicity and improve antifungal activity.     

Forty-channel phased array ultrasonic probe using 0.91Pb(Zn(1/3 )Nb(2/3))O(3)-0.09PbTiO(3) single crystal

A 0.91Pb(Zn(1/3)Nb(2/3))O(3)-0.09PbTiO (3) (PZN-PT) single crystal with high electromechanical coupling factor (k(33))>90% has been used to fabricate a 40-channel phased array ultrasonic probe with greater sensitivity and broader bandwidth than conventional probes. This probe has a center frequency of 3.5 MHz and an aperture of about 6.0x7.5 mm. The standard probe fabrication process was modified for PZN-PT. The dispersion of echo signals was within +/-20% of the mean value. After recovery poling, the echo amplitude of the PZN-PT single-crystal probe is 8 and 5 dB higher than that of one- and two-matching-layer PZT probes, respectively. Moreover, the fractional bandwidth of the single-matching-layer PZN-PT probe is broader than that of the two-matching-layer PZT probes. The PZN-PT single crystals provide great improvements in the sensitivity and bandwidth of phased array probes.     

A 20 MHz single-element ultrasonic probe using 0.91Pb(Zn(1/3 )Nb(2/3))O(3)-0.09PbTiO(3) single crystal

A 20 MHz single-element ultrasonic probe using 0.91Pb(Zn(1/3 )Nb(2/3))O(3)-0.09PbTiO(3) (PZN-PT 91/9) single crystal has been fabricated. The single crystal of PZN-PT 91/9 orientated to the (001) plane has longitudinal coupling factor of k(33)>90%, which is much larger than the k(33)=70 to 80% of conventional Pb(Zr(1-x),Ti(x))O(3) (PZT) based ceramics. A single crystal of PZN-PT 91/9 without inclusion or crack has been grown with dimensions of about 25x15x5 mm by the self-flux method. Because mechanical strength in the fabrication of disk transducers orientated to the (001) plane was sufficiently strong, under the same conditions as are applied to conventional PZT ceramics, a piston single-element probe with a diameter of 2.0 mm and a frequency of 20 MHz was successfully fabricated. The bandwidth of the PZN-PT 91/9 probe was 13-26 MHz, which was 4 MHz broader than that of the conventional PZT probe.     

Resistance to nonspecific protein adsorption by poly(vinyl alcohol) thin films adsorbed to a poly(styrene) support matrix studied using surface plasmon resonance.

Thin films of poly(vinyl alcohol) (PVA) polymer were prepared on a flat, nonporous, poly(styrene) support matrix by adsorption from aqueous solution and were characterized in order to investigate the nonspecific adsorption of proteins to a chromatographically relevant surface. The integrity and surface coverage of the PVA thin films were established by surface analysis and atomic force microscopy imaging. The adsorption of the PVA polymers to the poly(styrene) substrate and the nonspecific adsorption of proteins to the PVA-coated surface were monitored using surface plasmon resonance. PVA was strongly bound to the poly(styrene) surface, but the surface density of the adsorbed PVA polymers was affected substantially by the concentration, molecular weight, and degree of hydrolysis of PVA polymers used. There was evidence of increasing degrees of unfolding of the PVA polymer onto the poly(styrene) surface as the concentration of the the PVA coating solution increased. Complete PVA coverage of the poly(styrene) surface was observed at PVA concentrations of 0.1 mg/mL or greater but with significant influence of both molecular weight and degree of hydrolysis of the PVA polymers. Resistance of the PVA-coated poly(styrene) surface to the nonspecific adsorption of human serum albumin (HSA) correlated with the degree of surface coverage of the PVA. The use of anti-HSA as a probe for adsorbed HSA suggested that HSA was displacing PVA from the poly(styrene) surface at the lower PVA surface coverage. A complete barrier to nonspecific protein adsorption was observed with a PVA coating solution concentration of greater than 0.1 mg/ mL with a degree of hydrolysis of <88%.     

A 3.7 MHz phased array probe using 0.91Pb(Zn(1/3)Nb(2/3))O(3)-0.09PbTiO(3 )

A novel 128-channel phased array probe for echocardiography with a center frequency of 3.7 MHz using 0.91Pb(Zn(1/3)Nb(2/3))O(3)-0.09PbTiO(3 ) (PZN-9%PT) single crystal has been fabricated to realize greater sensitivity and broader bandwidth properties. The echo amplitude of the PZN-9%PT single-crystal probe is about 5 dB higher than that of the conventional lead airconate titanate (PZT) ceramic probe, and the fractional bandwidth is about 25 percentage points broader. The quality of B mode images obtained by the PZN-9%PT probe satisfies the performance of the two types of conventional PZT ceramic probes that have center frequencies of 2.5 and 3.75 MHz. At the reference frequency of 3 MHz, the Doppler sensitivity of the PZN-9%PT probe is about 5 dB higher than that of the 3.75 MHz PZT probe; the blood flow of a pulmonary vein in a hard-to-image patient is much more clearly imaged than in the case of using the PZT probe. These superior images are attributable to the use of sufficiently large PZN-9%PT single crystals obtained by the self-flux method.     

Spin polarization measurement of homogeneously doped Fe(1-x)Co(x)Si nanowires by Andreev reflection spectroscopy

We report a general method for determining the spin polarization from nanowire materials using Andreev reflection spectroscopy implemented with a Nb superconducting contact and common electron-beam lithography device fabrication techniques. This method was applied to magnetic semiconducting Fe(1-x)Co(x)Si alloy nanowires with x? = 0.23, and the average spin polarization extracted from 6 nanowire devices is 28 ± 7% with a highest observed value of 35%. Local-electrode atom probe tomography (APT) confirms the homogeneous distribution of Co atoms in the FeSi host lattice, and X-ray magnetic circular dichroism (XMCD) establishes that the elemental origin of magnetism in this strongly correlated electron system is due to Co atoms.     

Size dependent effects of antifungal phytogenic silver nanoparticles on germination,growth and biochemical parameters of rice (Oryza sativa L), maize (Zea mays L) and peanut (Arachis hypogaea L)

Advancement in materials synthesis largely depends up on their diverse applications and commercialisation. Antifungal effects of phytogenic silver nanoparticles (AgNPs) were evident, but the reports on the effects of the same on agricultural crops are scant. Herein, we report for the first time, size dependent effects of phytogenic AgNPs (synthesised using Stevia rebaudiana leaf extract) on the germination, growth and biochemical parameters of three important agricultural crops viz., rice (Oryza sativa L), maize (Zea mays L) and peanut (Arachis hypogaea L). AgNPs with varied sizes were prepared by changing the concentration and quantity of the Stevia rebaudiana leaf extract. As prepared AgNPs were characterized using the techniques, such as high‐resolution transmission electron microscopy, particle size and zeta potential analyser. The measured (dynamic light scattering technique) average sizes of particles are ranging from 68.5 to 116 nm. Fourier transform infrared studies confirmed the participation of alcohols, aldehydes and amides in the reduction and stabilisation of the AgNPs. Application of these AgNPs to three agricultural crop seeds (rice, maize and peanut) resulted in size dependent effects on their germination, growth and biochemical parameters such as, chlorophyll content, carotenoid and protein content. Further, antifungal activity of AgNPs also evaluated against fungi, Aspergillus niger.Inspec keywords: antibacterial activity, silver, nanoparticles, nanomedicine, biochemistry, crops, transmission electron microscopy, Fourier transform infrared spectra, electrokinetic effects, organic compounds, particle size, plant diseases, microorganismsOther keywords: fungi, Aspergillus niger, size 68.5 nm to 116 nm, silver particles, phytoextract, amide functional groups, aldehydes, alcohols, Fourier transform infrared studies, zeta potential analyser, particle size, high‐resolution transmission electron microscopy, Stevia plant leaf extract, agricultural crops, antifungal effects, materials synthesis, Arachis hypogaea L, peanut, Zea mays L, maize, Oryza sativa L, rice, biochemical parameters, growth, germination, antifungal phytogenic silver nanoparticles, size dependent effects     

Generation of ultralarge surface enhanced Raman spectroscopy (SERS)-active hot-spot volumes by an array of 2D nano-superlenses

Surface Enhanced Raman Spectroscopy (or SERS) has received tremendous attention in the past three decades. However, the extremely-confined probe volume (1 nm) of the plasmonic hot-spots occurring on a conventional roughened SERS-active metallic surface has limited value in macro-molecular studies. In this article, we show the plausibility of generating large SERS hot-spot volumes on an atomically-flat metal surface based upon a special 3D adiabatic plasmonic nano-focusing effect brought about by an array of nano-scale superlenses. We experimentally demonstrate the feasibility of this particular approach and report, for the first time, the acquisition of whole-protein SERS spectra of a layer of test protein, Cytochrome-c, using a custom-made Otto-Raman spectroscopy system equipped with nano-fluidics. Our study shows the potential of whole-protein SERS spectroscopy as a useful analytical tool that complements surface probe microscopies.