转移缓冲液对蛋白质印迹的影响

利用三种转移缓冲液,分别以半干和湿转法,检测癌基因产物Bcl-2, 并获得不同强度的印迹结果.不含SDS的转移缓冲液转移效果明显优于含SDS的转移缓冲液,且SDS含量越高印迹带越弱;此外,半干转移与湿转相比,可大大缩短转移时间,但转移效果不及湿转理想且稳定性较差.     

PRAS40(Ser183)磷酸化多克隆抗体的制备及检测

PRAS40为富含脯氨酸、分子量为40kD的Akt底物蛋白,能够与雷怕霉素哺乳动物细胞靶点复合物1(mTORC1)结合,其苏氨酸183位点(Ser183)可被mTORC1磷酸化。为了制备PRAS40(Ser183)磷酸化多克隆抗体,本实验通过蛋白疏水性抗原性分析设计多肽抗原,用其免疫家兔获得抗血清,ELISA检测其效价为1:10000;Western blotting法检测发现,通过rProtein A Sepharose亲和层析纯化并经非磷酸化的抗原条吸附处理后的抗体可以明显提高磷酸化抗体的特异性;用PRAS40抗体及PRAS40(Ser183)磷酸化抗体对正常细胞HL7702、HEK293及肿瘤细胞HepG2、A549、S180的检测显示:磷酸化的Ser183在不同细胞中表达差异不显著,而在经细胞饥饿处理的HEK293细胞中却明显观察到了S183磷酸化水平随氨基酸含量降低而减弱的现象。因此,本实验所制备的抗体可用于PRAS40(Ser183)磷酸化位点的功能研究。     

牛病毒性腹泻病毒E2抗原表位表达及验证

[目的]获得高效表达的重组牛病毒性腹泻病毒(Bovine viral diarrhoea virus,BVDV)E2免疫抗原蛋白。[方法]应用软件分析BVDV-E2蛋白抗原表位,将富含抗原表位基因片段连接表达载体p GEX-4T-1,并转化至大肠杆菌(DE3),经IPTG诱导表达,SDS-PAGE和Western Blot检测诱导表达蛋白,经镍柱分离纯化;以纯化蛋白为包被抗原,用间接ELISA检测抗原对免疫血清的反应性。[结果]成功克隆BVDV-E2抗原表位基因,并在大肠杆菌中高效表达,Western Blot和ELISA证实表达重组蛋白对BVDV阳性血清具有反应原性。[结论]获得大小为49.5 k Da的BVDV-E2抗原表位蛋白,并且该抗原稀释度在1:40,抗体稀释度在1:20时结合效果最佳。可用于后续的纳米抗体筛选的研究。     

黄瓜细菌性白枯病菌特异性抗体制备及其ELISA检测方法的建立

目的:制备黄瓜细菌性白枯病菌特异性抗体,建立其酶联免疫吸附分析(ELISA)的检测方法.方法:分别以黄瓜细菌性白枯病菌胞内蛋白和菌体为免疫原,制备两再种抗体,优化间接ELISA检测条件.结果:间接ELISA法测定两种纯化的菌体抗体(J4)和胞内蛋白抗体(P2)效价分别为1:32000和1:4000,二抗的最佳稀释度为1:3000,方阵试验表明J4的工作浓度为1:16000,抗原的最佳包被浓度为10<'6>cfu/ml,P2的工作浓度为1:2000,抗原的最佳包被浓度为10<'7>cfu/ml,两种抗体的灵敏度均为10<'5>cfu/ml,与黄瓜细菌性角斑病菌等26个菌株无交叉反应,特异性强.结论:成功制备了黄瓜细菌性白枯病菌特异性抗体,建立了ELISA检测方法.     

针对人Tudor-SN蛋白T103位点的应激磷酸化抗体制备及分析

目的：针对人Tudor-SN蛋白T103位点（103位苏氨酸,Thr103）制备兔源多克隆磷酸化抗体,并进行应激磷酸化的时相分析。方法：首先人工合成含磷酸化T103（pT103）位点的多肽,4次免疫新西兰大白兔后获取抗血清;然后以AKTA蛋白纯化系统进行纯化,并利用Western blotting和细胞免疫荧光实验对纯化后的抗pT103抗体进行鉴定;最后以In-cell Western法进行Tudor-SN蛋白的应激磷酸化/去磷酸化时相性分析。结果：（1）确定并合成磷酸化多肽“TIENKpTPQGRC”,收集约75 ml兔源抗血清,纯化后获取2.08 mg/ml抗pT103抗体;（2）当HeLa细胞受到氧化应激时,以pT103抗体检测的磷酸化信号增强,可在胞浆中检测到颗粒状信号,与内源性Tudor-SN应激颗粒存在共定位关系;（3）在氧化应激及应激去除后恢复过程中,T103位点的磷酸化水平呈现一定的波动性时相。结论：成功制备针对Tudor-SN蛋白T103位点的兔源多克隆抗pT103抗体,有助于从磷酸化修饰角度进行Tudor-SN在细胞应激方面的机制探讨。     

布鲁菌核糖体蛋白L7/L12的表达纯化及生物活性鉴定

目的:原核表达系统表达布鲁菌核糖体蛋白L7/L12与GST的融合蛋白GST-L7/L12,并纯化蛋白L7/L12,建立检测特异性抗体的间接ELISA方法。方法:对含有L7/L12的原核表达载体pGEX-4T-1-L7/L12进行了原核表达。利用亲和层析柱分别纯化融合蛋白GST-L7/L12和蛋白L7/L12,并用SDS-PAGE及Western印迹分析鉴定。以L7/L12为抗原包被微量板,优化抗原包被浓度和羊抗鼠IgG-HRP稀释度,建立间接ELISA方法,并检测其特异性。结果:SDS-PAGE结果显示在相对分子质量为38000和12000处可见纯化蛋白的条带,Western印迹分析表明这2条带均能被免疫兔血清识别,表明获得了纯化的有生物活性的融合蛋白GST-L7/L12和蛋白L7/L12。间接ELISA方法的L7/L12抗原包被浓度为5μg/mL,羊抗鼠酶标二抗稀释度为1∶1000。小鼠免疫血清与L7/L12抗原出现阳性反应,而与布鲁菌融合蛋白OMP31、结核分枝杆菌抗原85b及牛血清白蛋白则呈阴性。结论:成功地对布鲁菌核糖体蛋白L7/L12进行了原核表达和纯化,以其为基础建立的间接ELISA方法稳定且特异。     

间接竞争ELISA法对柞蚕微孢子虫的检测

柞蚕微孢子虫病是柞蚕唯一的检疫性病害,其致病病原物为柞蚕微孢子虫(Nosema pernyi Ding,Su&Wen),因此,柞蚕微孢子虫的检测对于该病的防治具有重要意义。本文通过制备柞蚕微孢子虫多克隆抗体,建立柞蚕微孢子虫间接竞争ELISA检测法。结果表明,柞蚕微孢子虫多克隆抗体效价为1∶104、浓度为3 mg·mL-1,主要由2条大小约50 ku和25 ku蛋白条带组成,可作为后续试验多克隆抗体材料。间接竞争ELISA法最佳抗原工作浓度为2.0μg·mL-1微孢子虫孢壁蛋白溶液,最佳抗体工作浓度为兔抗血清按1∶102倍浓度稀释,酶标二抗最佳工作浓度为1∶5×104倍稀释,柞蚕微孢子虫间接竞争ELISA检测法的灵敏度为1.6×105spores·mL-1。间接竞争ELISA法在柞蚕微孢子虫的检测方面具有一定的应用价值。     

小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法的建立

本研究旨在建立小反刍兽疫病毒H蛋白抗体的化学发光免疫分析检测方法。以H蛋白4个反应原性较好的B细胞表位串联后为检测抗原,在确定抗原包被量和血清稀释度,优化血清和酶标二抗反应时间的基础上,用ROC曲线分析确定检测临界值,建立了小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法,然后对该方法进行敏感性、特异性和重复性评价。结果显示,建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法最佳抗原包被浓度为1.5×10^-6μg/孔,待检血清最佳稀释度为1∶100;血清及酶标二抗孵育时间均为10 min,检测方法的临界值为S/P=9.77%,批内及批间变异系数均小于10%;与口蹄疫、羊痘、蓝舌病及山羊传染性胸膜肺炎阳性血清不发生交叉反应;用建立的化学发光法和市售小反刍兽疫抗体cELISA试剂盒平行检测247份田间血清,两者相对符合率为94.33%,但化学发光法敏感性更高。研究结果表明,建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析方法灵敏、特异、稳定,可用于田间血清样品小反刍兽疫抗体检测。     

甘蔗高粱花叶病毒间接ELISA检测方法的建立和应用

利用人工合成的高粱花叶病毒P3蛋白的多肽片段作为抗原,制备多克隆抗体,利用免疫印迹对抗体的特异性进行鉴定,表明抗体具有高度特异性。通过方阵滴定法确定抗原和抗体的最佳工作浓度,并分别对包被时间、二抗工作浓度、孵育条件、底物显色时间等进行优化,从而建立了高粱花叶病毒(Sr MV)的间接ELISA高通量检测方法。通过测试确定了阴阳性结果判定的临界值,评估了该方法的灵敏度以及与RT-PCR检测法的符合度。所建立的间接ELISA检测方法的抗原、一抗、二抗最佳稀释度分别为1:4、1:600和1:5 000;最佳抗原包被条件为4℃、12 h;抗原抗体最佳孵育条件为37℃、60 min;二抗最佳孵育条件为37℃、45 min;最佳显色条件为37℃、12 min;此检测方法检测速度快、灵敏度高、特异性强,与RT-PCR法的符合度为87.00%。     

严重急性呼吸综合征患者血清特异性抗体消涨规律的长期追踪研究

目的:追踪检测SARS冠状病毒(SARS-CoV)抗体在严重急性呼吸综合征(SARS)患者血清中的产生及其转归规律,为SARS诊断及防治提供依据。方法:对41例临床诊断SARS患者的血清进行了连续3年的检测,分别应用间接免疫荧光(IFA)检测患者血清特异性IgG抗体平均滴度,应用双抗原夹心ELISA法检测患者血清核衣壳蛋白(N蛋白)抗体的平均滴度,绘制消涨曲线,得出消涨规律。结果:应用IFA检测患者血清特异性IgG抗体与应用双抗原夹心ELISA法检测N蛋白抗体所得到的消涨规律不同,前者测得康复者血清IgG抗体滴度维持在较低水平,但后者检测35例康复者血清N蛋白抗体仍维持在较高水平。结论:SARS-CoV的N蛋白是免疫原性较强的抗原,感染3年后仍存在高滴度抗体;抗原夹心ELISA检测SARS-CoV N蛋白抗体的灵敏度较IFA方法高。     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

注射用转移因子不同生产方法比较

比较从猪脾中提取活性转移因子的不同生产方法,以建立适合产业化的制造工艺。将猪脾匀浆后,分别冻融0、3、5次,再以超滤法或Lawence法提取转移因子。与lawrence法相比,经5次冻融并超滤制备的产品收率高,符合国家标准。匀浆冻融次数对转移因子的收率和质量有一定的影响,超滤法适合于转移因子的大规模生产。     

Determination of metallic ion transfer from an implanted prosthesis by the PIXE method

Prostheses can release some metallic elements to the surrounding tissues, particularly when they are not covered with a biomaterial layer and when an unsealing process happens. We try to measure major and trace elements in these tissues with an experimentally sensitive method. Proton-induced X-ray emission is used to detect about 10 elements in tissue. Tissues are calcinated and deposited in a thin layer before irradiation. Results are obtained in a standard and samples from three patients. We observe contamination by Ti, Cr, Ni, and Zn in the tissues. Correlations are to be studied between these atomic transfers and prosthesis in the patient.     

Critical assessment of gassing-in methods for measuring k(l)a in fermentors

The reliability of dynamic measurement methods of k(l)a in fermentors using a step oxygen concentration change in the feed gas was tested. The tests were performed both for the original variant using the nitrogen right harpoon over left harpoon air exchange and the newly presented variant using the oxygen-enriched air (27 vol % O(2)) --> air exchange. The testing consisted in comparing k(l)a values determined from these methods with values determined from the steady-state Na(2)SO(3) feeding method and the dynamic pressure method, the reliability of which was proven earlier. The measurements were done in water (coalescent batch) and in 0.5M Na(2)SO(4) solution with and without the addition of 1 wt % carboxymethylcellulose (noncoalescent batches). It was found that in noncoalescent liquids the methods tested give extremely low k(l)a values (as low as 15% of the correct value). The methods are defective in principle irrespective of the gases used for exchange.     

Impact of bio-nanogold on seed germination and seedling growth in Pennisetum glaucum

Nanotechnology is leading towards the development of low cost applications to improve the cultivation and growth of plants. The use of nanotechnology in agriculture will leads to a significant effect on food industry along with opening a new area of research in agroecosystem. In this paper gold nanoparticles were biosynthesized with Cassia auriculata leaf extract at room temperature and characterized by UV–vis spectroscopy, X-ray diffraction and transmission electron microscopy. The objective of this study was to investigate effect of synthesized bio-nanogold on an important food and biofuel producing plant Pennisetum glaucum. Positive effects were observed on percentage of seed germination and growth of seedlings. Improved germination and increased plant biomass have high economic importance in production of biofuel or raw materials, agriculture and horticulture. Although the impact of nanoparticles on plants depends on concentration, size and shape. The biological synthesized AuNPs can replace the chemically synthesized AuNPs used in gene transfer method. The study gives brief insight on nanoparticles effects on plants, brings attention on both positive and negative side of nanomaterial which can resolve phytopathological infections by stimulating nutrition and growth.     

Efficient fluorescence energy transfer system between fluorescein isothiocyanate and CdTe quantum dots for the detection of silver ions

We report a fluorescence resonance energy transfer (FRET) system in which the fluorescent donor is fluorescein isothiocyanate (FITC) dye and the fluorescent acceptor is CdTe quantum dot (QDs). Based on FRET quenching theory, we designed a method to detect the concentration of silver ions (Ag⁺). The results revealed a good linear trend over Ag⁺ concentrations in the range 0.01–8.96 nmol/L, a range that was larger than with other methods; the quenching coefficient is 0.442. The FRET mechanism and physical mechanisms responsible for dynamic quenching are also discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

Pathways for Electron Tunneling in Cytochrome c Oxidase

Warburg showed in 1929 that the photochemical action spectrum for CO dissociation from cytochrome c oxidase is that of a heme protein. Keilin had shown that cytochrome a does not react with oxygen, so he did not accept Warburg's view until 1939, when he discovered cytochrome a ₃. The dinuclear cytochrome a ₃-Cu_B unit was found by EPR in 1967, whereas the dinuclear nature of the Cu_A site was not universally accepted until oxidase crystal structures were published in 1995. There are negative redox interactions between cytochrome a and the other redox sites in the oxidase, so that the reduction potential of a particular site depends on the redox states of the other sites. Calculated electron-tunneling pathways for internal electron transfer in the oxidase indicate that the coupling-limited rates are 9×10⁵ (Cu _A a) and 7×10⁶ s^–1 (a a ₃); these calculations are in reasonable agreement with experimental rates, after corrections are made for driving force and reorganization energy. The best Cu_A-a pathway starts from the ligand His204 and not from the bridging sulfur of Cys196, and an efficient a-a ₃ path involves the heme ligands His378 and His376 as well as the intervening Phe377 residue. All direct paths from Cu_A to a ₃ are poor, indicating that direct Cu_A a ₃ electron transfer is much slower than the Cu_A a reaction. The pathways model suggests a means for gating the electron flow in redox-linked proton pumps.     

Proton Transfer in Liquid Water II; A Semiempirical Method to Describe Chemical Reactions

Abstract

A new semiempirical method is developed to deal with the proton transfer in liquid water. In the previous work, we have shown that two- and three-body charge transfer interactions and electrostatic interactions are the most important factors to describe the potential energy surfaces (PES) of the proton transfer in liquid water [Chemical Physics 180, 239–269, 1994], In order to take account of these factors, we develop a semiempirical method imposing the principle of electronegativity equalization to the Atoms in Molecule (AIM) method. The method is free from the well-known discrepancy of the traditional AIM methods, that is, the fractional molecular charges at large molecular separation, and thus can be applied to the charge transfer reactions. Intra- and intermolecular physical quantities, such as total energies, force vectors, dipole moment vectors and intermolecular charge transfer, obtained by the present method are found to be in good agreement with those by ab initio calculation.     

Combined sulfite method for the measurement of the oxygen transfer coefficient k(L)a in bio-reactors

The combined sulfite method is proposed for the measurement of oxygen transfer coefficients, k_La, in bioreactors. The method consists of a steady-state and a dynamic measurement which are carried out under the same experimental conditions and thus yield data for both methods during one experiment. The applied experimental conditions are shown to avoid chemical enhancement during the steady-state measurement. Moreover, no parallel sulfite oxidation occurs during the oxygen saturation phase of the dynamic measurement. Under the applied experimental conditions, no information about the sulfite oxidation kinetics is required and possible metal ion impurities in sulfite salts do not influence the measurement. The characterization of a laboratory-scale bioreactor aerated with pure oxygen yields k_La values during the steady-state and the dynamic measurements that are in good agreement with the dynamic pressure method, the correctness of which is generally accepted. When air is used for absorption, the steady-state measurement yields k_La values that correlate to the correct variant of the standard dynamic method. The dynamic measurement with air absorption yields a k_La value which considers the influence of the non-uniform bubble size distribution present in bubble-aerated bioreactors.