蛋白芯片生产用醛基化玻片制备的条件优化及初步检定

建立一种以玻片为基质的蛋白芯片制备的优化及检定方法。将经过清洗处理的玻片,用不同浓度的氨基硅烷试剂和戊二醛试剂,在不同的时间内,进行氨基化和醛基化处理,通过蛋白结合强度与处理时间及溶剂浓度的动力学相关性分析,确定最佳的优化条件,将不同浓度梯度的人IgG抗体结合于该玻片表面,经过洗涤、封闭,再加入Cy3荧光标记的二抗,孵育,洗涤后检测各点的荧光强度,确定其线性范围及其检测灵敏度。氨基硅烷试剂浓度为5%,作用时间为30min;戊二醛试剂浓度为2.5%,作用时间为60min时,蛋白结合强度达到饱和。蛋白芯片在2～2×4-7mg/ml浓度范围内有良好的线性,能够检测出人IgG的最低浓度为2×4-8mg/ml。     

Intracellular recordings from nonspiking interneurons in a semiintact,tethered walking insect

Nonspiking interneurons were investigated in a tethered, walking insect, Carausius morosus, that was able to freely perform walking movements. Experiments were carried out with animals walking on a lightweight, double-wheel treadmill. Although the animal was opened dorsally, the walking system was left intact. Intracellular recordings were obtained from the dorsal posterior neuropil of the mesothoracic ganglion. Nonspiking inter-neurons, in which modulations of the membrane potential were correlated with the walking rhythm, were described physiologically and stained with Lucifer Yellow. Interneurons are demonstrated in which membrane potential oscillations mirror the leg position or show correlation with the motoneuronal activity of the protractor and retractor coxae muscles during walking. Other interneurons showed distinct hyperpolarizations at certain important trigger points in the step cycle, for example, at the extreme posterior position. Through electrical stimulation of single, nonspiking interneurons during walking, the motoneuronal activity in two antagonistic muscles—protractor and retractor coxae—could be reversed and even the movement of the ipsilateral leg could be influenced. The nonspiking interneurons described appear to be important premotor elements involved in walking. They receive, integrate, and process information from different leg proprioceptors and drive groups of leg motoneurons during walking.     

人高迁移率族蛋白B1，高迁移率族蛋白B1 A box和B box的原核表达、 纯化及高迁移率族蛋白B1多克隆抗血清的制备

目的:获取重组人高迁移率族蛋白B1(HMGB1),HMGB1Abox和Bbox的纯化蛋白,制备HMGB1的多克隆抗血清。方法:采用PCR方法扩增人HMGB1,HMGB1的Abox和Bbox目的基因片段,构建原核表达载体,进行原核表达与蛋白纯化,然后用HMGB1免疫新西兰大白兔,制备多克隆抗血清。采用ELISA检测抗血清效价,用免疫组化检测HMGB1在小鼠肝损伤组织中的表达。结果:成功构建了人HMGB1,HMGB1的Abox和Bbox原核表达载体pET28-HMGB1、pET28-Abox、pET28-Bbox,在E.coli BL21中表达,镍亲和层析柱提纯,获取纯净目的蛋白。HMGB1免疫新西兰大白兔后,抗血清效价为1:2,000,000,具有高度特异性。免疫组化显示小鼠坏死肝组织HMGB1表达增加。结论:本研究获得了人HMGB1以及HMGB1的Abox和Bbox的纯化蛋白,制备了人HMGB1的多克隆抗血清,为HMGB1的结构、组织表达谱及其功能的研究奠定了基础。     

Effective field trips in nature: the interplay between novelty and learning

Educational field trips are common practice in environmental education and education for sustainable development, well recognised by researchers for their potential to achieve cognitive and affective educational outcomes. One of the factors that influences learning during field trips is their novelty. The current study focuses on the interplay between novelty, preparation and environmental learning outcomes of 5th and 6th grade students during a typical field trip in Flanders. Our dependent variables are Inclusion of Nature in the Self, the two major ecological values Preservation and Utilisation and ecosystem knowledge. The sample includes 484 students (10–12 years old) and their 24 teachers. Key questions addressed are: (1) What is learned during the field trip? (2) What is the level of novelty for students during a field trip? (3) How does the novelty effect relate to learning? Results show that participation in the field trip leads to a substantial increase in ecosystem knowledge, but fails in reaching the affective goals set out by the field trip organisers. Our results furthermore provide support for the hypothesised non-linear relationship between novelty and knowledge gain, showing that while a little novelty is positive, too much novelty can stand in the way of learning.     

A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96‐well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on‐bead digested by using Single‐Pot solid‐phase sample preparation (SP3). The whole IP‐SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter‐aided sample preparation) or a longer incubation protocol. Taken together, the IP‐SP3 protocol is a fast and economical approach easily applicable for large‐scale protein interactome analysis.     

蛋白质组学研究相关技术及进展

蛋白质组学以蛋白质组为研究对象,应用相关研究技术,从整体水平上来认识蛋白的存在及活动方式。随着人类基因组计划的完成,蛋白质组学的研究也得到了快速发展,与蛋白质组学研究相关的一些技术也日益得到完善和提高。简要综述了近年来蛋白质组学研究中最为重要的样品制备、蛋白质分离、蛋白质鉴定等技术及研究进展。     

秸秆降解菌剂对秸秆还田土壤中细菌种群数量的影响

要研究秸秆降解菌剂施用后1个生长季秸秆中细菌的数量和纤维素酶活力,了解其动态变化,为进一步研究分析土壤中微生物结构变化规律提供可靠的理论依据.利用分子生物学方法,对秸秆降解菌剂施用后不同生长阶段耕层中细菌基因组DNA纯化后PCR扩增其16S rDNA V3可变区,对扩增产物进行DGGE电泳,并对结果进行分析.初步分析表明,菌落数量呈现前期上升、中期最高、后期逐渐降低的趋势.     

EZYprep LC‐coupled MALDI‐TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.     

Sampling dust from human dwellings to estimate the prevalence ofDermatophagoides mite and cat allergens

Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens.     

北虫草复合制剂对小鼠免疫功能的影响

探讨北虫草复合制剂对小鼠免疫功能的调节作用。建立小鼠免疫力降低的动物模型,实验分6组:对照组、衰老/免疫抑制模型组、白介素-2(IL-2)和北虫草复合制剂的不同剂量组。采用称重法测定免疫器官重量,计算胸腺指数和脾指数。小鼠溶血素抗体生成采用绵羊红细胞致敏法。北虫草复合制剂对小鼠脾脏指数和胸腺指数的影响:实验组(50.2±2.4与27.6±3.6)明显高于模型组(45.6±4.8与23.6±3.6),单位:(mg/10 g体重),差异有显著性(P<0.05)。对小鼠溶血素抗体生成的影响:实验组(53.53±7.8)高于药物对照组(36.50±7.3)。北虫草复合制剂能恢复衰老小鼠的胸腺指数和脾脏指数,增强免疫抑制小鼠血清溶血素含量,因此,对免疫功能低下的小鼠模型具有免疫调节作用。