59 resultados para Two-Hybrid System Techniques

em Chinese Academy of Sciences Institutional Repositories Grid Portal


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In this paper, we attempt to develop a sensitive detection method for glucose with the combination of the unique optical property of quantum dots and the specificity of enzymatic reactions. With glucose and hydroquinone as substrates, benzoquinone that intensively quenches the photoluminescence of quantum dots can be produced via the catalysis of bienzyme (glucose oxidase and horseradish peroxidase) system. A relatively low detection limit of 1.0 x 10(-8) mol/L can be achieved. Two linear ranges from 1.0 x 10(-6) to 1.5 x 10(-4) M and from 1.5 x 10(-4) to 1.0 x 10(-3) M were obtained.

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Classical theories have successfully provided an explanation for convection in a liquid layer heated from below without evaporation. However, these theories are inadequate to account for the convective instabilities in an evaporating liquid layer, especially in the case when it is cooled from below. In the present paper, we study the onset of Marangoni convection in a liquid layer being overlain by a vapor layer.A new two-sided model is put forward instead of the one-sided model in previous studies. Marangoni-Bénard instabilities in evaporating liquid thin layers are investigated with a linear instability analysis. We define a new evaporation Biot number, which is different from that in previous studies and discuss the influences of reference evaporating velocity and evaporation Biot number on the vapor-liquid system. At the end, we explain why the instability occurs even when an evaporating liquid layer is cooled from below.

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This paper deals with the interaction of solitary waves in a two-fluid system which consistsof two superimposed incompressible inviscid fluids with a free surface and a horizontal rigidbottom. Under the assumption of shallow water wave, we first derive the basic equationssuitable for the model considered, a generalized form of the Boussinesq equations, then usingthe PLK method and the reductive perturbation method, obtain the second-order approximatesolution for the head-on collision between two pairs of interface and surface solitary waves,and give their maximum amplitudes during the collision and the nonuniform phase shiftsafter the collision which lead to the distortion of the wave profiles.

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The convective instabilities in two or more superposed layers heated from below were studied extensively by many scientists due to several interfacial phenomena in nature and crystal growth application. Most works of them were performed mainly on the instability behaviors induced only by buoyancy force, especially on the oscillatory behavior at onset of convection (see Gershuni et. Al.(1982), Renardy et. Al. (1985,2000), Rasenat et. Al. (1989), and Colinet et. Al.(1994)) . But the unstable situations of multi-layer liquid convection will become more complicated and interesting while considering at the same time the buoyancy effect combined with thermocapillary effect. This is the case in the gravity reduced field or thin liquid layer where the thermocapillary effect is as important as buoyancy effect. The objective of this study was to investigate theoretically the interaction between Rayleigh-Bénard instability and pure Marangoni instability in a two-layer system, and more attention focus on the oscillatory instability both at the onset of convection and with increasing supercriticality. Oscillatory behavious of Rayleigh-Marangoni-Bénard convective instability (R-M-B instability) and flow patterns are presented in the two-layer system of Silicon Oil (10cSt) over Fluorinert (FC70) for a larger various range of two-layer depth ratios (Hr=Hupper/Hdown) from 0.2 to 5.0. Both linear instability analysis and 2D numerical simulation (A=L/H=10) show that the instability of the system depends strongly on the depth ratio of two-layer liquids. The oscillatory instability regime at the onset of R-M-B convection are found theoretically in different regions of layer thickness ratio for different two-layer depth H=12,6,4,3mm. The neutral stability curve of the system displaces to right while we consider the Marangoni effect at the interface in comparison with the Rayleigh-Bénard instability of the system without the Marangoni effect (Ma=0). The numerical results show different regimes of the developing of convection in the two-layer system for different thickness ratios and some differences at the onset of pure Marangoni convection and the onset of Rayleigh-Bénard convections in two-layer liquids. Both traveling wave and standing wave were detected in the oscillatory instability regime due to the competition between Rayleigh-Bénard instability and Marangoni effect. The mechanism of the standing wave formation in the system is presented numerically in this paper. The oscillating standing wave results in the competition of the intermediate Marangoni cell and the Rayleigh convective rolls. In the two-layer system of 47v2 silicone oil over water, a transition form the steady instability to the oscillatory instability of the Rayleigh-Marangoni-Bénard Convection was found numerically above the onset of convection for ε=0.9 and Hr=0.5. We propose that this oscillatory mechanism is possible to explain the experimental observation of Degen et. Al.(1998). Experimental work in comparison with our theoretical findings on the two-layer Rayleigh-Marangoni-Bénard convection with thinner depth for H<6mm will be carried out in the near future, and more attention will be paid to new oscillatory instability regimes possible in the influence of thermocapillary effects on the competition of two-layer liquids

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Spin dynamics in the first and second subbands have been examined simultaneously by time resolved Kerr rotation in a single-barrier heterostructure of a 500 nm thick GaAs absorption layer. By scanning the wavelengths of the probe and pump beams towards the short wavelength in the zero magnetic field, the spin coherent time T-2(1)* in the 1st subband E-1 decreases in accordance with the D'yakonov-Perel' (DP) spin decoherence mechanism. Meanwhile, the spin coherence time T-2(2)* in the 2nd subband E-2 remains very low at wavelengths longer than 810 nm, and then is dramatically enhanced afterwards. At 803 nm, T-2(2)* (450 ps) becomes ten times longer than T-2(1)* (50 ps). A new feature has been discovered at the wavelength of 811nm under the bias of -0.3V (807nm under the bias of -0.6V) that the spin coherence times (T-2(1)* and T-2(2)*) and the effective g* factors (vertical bar g*(E-1)vertical bar and vertical bar g*(E-2)vertical bar) all display a sudden change, presumably due to the "resonant" spin exchange coupling between two spin opposite bands. Copyright (C) EPLA, 2008.

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The thermal entanglement in a two-qutrit system with two spins coupled by exchange interaction is investigated in terms of the measure of entanglement called "negativity". It is found that the thermal entanglement is present and evolvements symmetrically between both ferromagnetic and antiferromagnetic exchange couplings with the temperature. Moreover the critical temperature at which the negativity vanishes increases with the exchange coupling constant J. From the temperature and magnetic field dependences we demonstrate that the temperature and the magnetic field can affect the feature of the thermal entanglement significantly.

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A complete set of match calculation methods for optimum sizing of PV/wind hybrid system is presented. In this method, the more accurate and practical mathematic models for characterizing PV module, wind generator and battery are adopted; combining with hourly measured meteorologic data and load data, the performance of a PV/wind hybrid system is determined on a hourly basis; by fixing the capacity of wind generators, the whole year's LPSP (loss of power supply probability) values of PV/wind hybrid systems with different capacity of PV array and battery bank are calculated, then the trade-off curve between battery bank and PV array capacity is drawn for the given LPSP value; the optimum configuration which can meet the energy demand with the minimum cost can be found by drawing a tangent to the trade-off curve with the slope representing the relationship between cost of PV module and that of the battery. According to this match calculation method, a set of match calculation programs for optimum sizing of PV/wind hybrid systems have been developed. Applying these match calculation programs to an assumed PV/wind hybrid system to be installed at Waglan island of Hong Kong, the optimum configuration and its hourly, daily, monthly and yearly performances are given. (C) 2003 Elsevier Science Ltd. All rights reserved.

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人类的载脂蛋白A5(apolipoprotein A5,APOA5)是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级,但能显著影响血浆三酰甘油水平,对血脂代谢具有重要意义,可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低,直接从血浆中分离纯化很困难,国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性,探讨其与TG代谢中的其它关键成分之间的相互关系,揭示其在脂类代谢相关疾病中的重要地位,必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术,诱导表达纯化APOA5蛋白,免疫动物制备多克隆抗体,为进一步研究人肝脏细胞中APOA5的相互作用蛋白,研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能,我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术,免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系,为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术,从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD,构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21(DE3),成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列,用镍离子亲和柱进行纯化和复性后,获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔,获得了高效价的兔抗人APOA5多克隆抗体,Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒,经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功,并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白,与预测的分子量APOA5(41 kD)/lamda cI (27 kD)一致。自激活实验证明诱饵蛋白不能单独激活报告基因,可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选,分离出10个阳性克隆。对结果进行生物信息学分析,得到7个与APOA5相互作用的蛋白,其中BI1为细胞凋亡调节因子;ATP6、CYTB、ND2、COX-1为线粒体表达蛋白; ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1,利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5,并验证其表达。通过免疫荧光细胞内共定位研究发现,靶蛋白APOA5主要分布于胞浆,与BI1在HEK293细胞有共定位,即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段,在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果,为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

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The nucleation and growth kinetics of CdS nanocrystals in a two-phase synthesis system have been investigated. It was found that the nucleation process is quite lengthy and overlapped with the growth process; nevertheless, as formed nanocrystals show extremely narrow size distribution owing to the unique heterogeneous reacting environment and Ostwald ripening growth. The nucleation and growth kinetics of the nanocrystals were also influenced strongly by the monomer concentration, capping agent concentration, and solvent polarity. It was also found that a high monomer concentration, a low capping agent concentration, and low solvent polarity lead to a higher maximum nucleus concentration and nanocrystal concentration, while high polarity solvents are favorable for the formation of nanocrystals with narrower size distribution and higher photoluminescence quantum yield.

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In this paper, we attempt to construct a simple and sensitive detection method for both phenolic compounds and hydrogen peroxide, with the successful combination of the unique property of quantum dots and the specificity of enzymatic reactions. In the presence Of H2O2 and horseradish peroxidase, phenolic compounds can quench quantum dots' photoluminescence efficiently, and the extent of quenching is severalfold to more than 100-fold increase. Quinone intermediates produced from the enzymatic catalyzed oxidation of phenolic compounds were believed to play the main role in the photoluminescence quenching.

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poly(epsilon-caprolactone) (PCL) and silica (SiO2) organic-inorganic hybrid materials have been synthesized by sol-gel approach and the crystalline behavior of PCL in the silica networks has been investigated by differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD). The degree of PCL crystallinity in the PCL/SiO2 hybrid networks reduces with the increase of SiO2 content. PCL is in an amorphous state when the concentration of PCL is lower than 40wt% in the hybrid materials. The melting points of PCL in the networks are lower than that of pure PCL,but they almost have a same value. WAXD results show that when the PCL weight percentage is higher than 40wt% in the hybrid samples,part of PCL can crysatllize and the PCL crystallites are almost in a same size. That means the crystalline movement of PCL molecular chains is strictly confined by the porous gel. The crystalline PCL in the hybrid samples is relatively free from the composition of the materials, because the crystallization temperature and melting point of PCL of the samples are almost equal,and the crystalline PCL of different samples has the same crystalline structure and the same crystallite sizes L-110 and L-200, that means the crystalline part of PCL in the hybrid samples is unperturbed and the porous silica gel gives enough space for PCL to crystallize into the same crystalline structure and the same size crystallites.

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In the investigation of effect of KSCN on the partitioning of lysozyme in PEG2000/ammonium sulfate aqueous two-phase system, it was found that the KSCN could alter the pH difference between the two phases. and thus affect the partition of lysozyme. The relationship between partition coefficients of lysozyme and pH differences between two phases was discussed.

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The effect of potassium thiocyanate on the partitioning of lysozyme and BSA in polyethylene glycol 2000/ammonium sulfate aqueous two-phase system has been investigated. As a result of the addition of potassium thiocyanate to the PEG/ammonium sulfate system, the PEG/mixed salts aqueous two-phase system was formed. It was found that the potassium thiocyanate could alter the pH difference between the two phases, and, thus, influence the partition coefficients of the differently charged proteins. The relationship between partition coefficient of the proteins and pH difference between two phases has been discussed. It was proposed that the pH difference between two phases could be employed as the measurement of electrostatic driving force for the partitioning of charged proteins in polyethylene glycol 2000/ammonium sulfate aqueous two-phase system.

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Interfacial waves propagating along the interface between a three-dimensional two-fluid system with a rigid upper boundary and an uneven bottom are considered. There is a light fluid layer overlying a heavier one in the system, and a small density difference exists between the two layers. A set of higher-order Boussinesq-type equations in terms of the depth-averaged velocities accounting for stronger nonlinearity are derived. When the small parameter measuring frequency dispersion keeping up to lower-order and full nonlinearity are considered, the equations include the Choi and Camassa's results (1999). The enhanced equations in terms of the depth-averaged velocities are obtained by applying the enhancement technique introduced by Madsen et al. (1991) and Schaffer and Madsen (1995a). It is noted that the equations derived from the present study include, as special cases, those obtained by Madsen and Schaffer (1998). By comparison with the dispersion relation of the linear Stokes waves, we found that the dispersion relation is more improved than Choi and Camassa's (1999) results, and the applicable scope of water depth is deeper.