壳聚糖载体的制备及脲酶的固定化研究

以甲壳素为原料,制备出壳聚糖载体,并对脲酶进行固定化。通过测量悬挂醛基探讨了交联条件对载体性能的影响,优化了脲酶的固定化条件,研究了固定化酶的酶学性质,并与游离酶进行了比较。结果表明。制备载体的最优条件是将微球用6％的戊二醛活化2h,最佳联酶条件是载体与脲酶共反应1h。该固定化酶的最适温度为65℃,最适pH值为6．6,米氏常数为0．009mol／L,较游离酶均有较大改善。热稳定性较游离酶有很大的提高,且具有良好的操作稳定性。     

一种测定鳞翅目幼虫取食选择的方法——叶碟法及其改进和注意事项

叶碟法是鳞翅目幼虫取食选择实验常用的一个方法。文章以经典的Jermy叶碟法为基础,介绍对其的改进以及实验中应该注意的事项,如幼虫的选择、幼虫取食的持续时间、叶碟的数目以及叶碟面积的测量等。     

Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips

Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay.     

管式磁微粒非洲猪瘟病毒pp62蛋白化学发光抗体检测方法的建立

【背景】非洲猪瘟(African swine fever, ASF)作为高致病性传染病,给我国生猪养殖业造成了严重的经济损失,因此建立快捷、灵敏的诊断方法至关重要。【目的】建立一种管式非洲猪瘟病毒pp62蛋白化学发光抗体检测方法。【方法】以重组pp62蛋白作为包被抗原,羧基磁珠(carboxylic magnetic beads)作为固相载体,碱性磷酸酶(alkaline phosphatase, AP)标记的兔抗猪IgG作为酶标二抗,通过对反应条件进行优化,采用非洲猪瘟国家参考品作为溯源血清绘制出标准曲线,建立基于ASFV pp62蛋白的化学发光抗体检测方法。【结果】用建立的化学发光方法检测277份临床样品血清,通过受试者工作特征(receiver operating characteristic, ROC)曲线分析确定阴性、阳性临界值,并确定判定标准：浓度值>140.02 U时为抗体阳性,浓度值<140.02 U时为抗体阴性。此方法对6种不同的病原血清抗体进行检测均无交叉反应,且批内和批间变异系数均在10%以内,与商品化非洲猪瘟抗体检测试剂盒的符合率达96.7%。【结论】本研究建立的管式非洲猪瘟病毒化学发光抗体检测方法具有良好的特异性、敏感性和重复性,可为非洲猪瘟早期监测及试剂盒研发提供参考。     

一种改进的丝状真菌DNA提取方法

以丝状真菌雅致枝霉(Thamnidium elegans)和深黄伞形霉(Umbelopsis isabellina)为材料,使用优化的CTAB法提取基因组DNA.改进后的方法使用液氮冻融以及玻璃珠振荡的方法代替了传统的液氮研磨,所需茵体量少,而得到的基因组DNA比用传统的CTAB法得到的基因组DNA产率高,纯度好、且步骤简单,适用于一次微量提取多个样品的基因组DNA.这种方法得到的基因组DNA可用于大部分分子生物学基本实验如PCR和DNA的酶切等.     

Preservation of the chondrocyte's pericellular matrix improves cell‐induced cartilage formation

The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real‐time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross‐linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50‐fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell‐induced cartilage production. J. Cell. Biochem. 110: 260–271, 2010. © 2010 Wiley‐Liss, Inc.     

Differences in colonisation of five marine bacteria on two types of glass surfaces

The retention patterns of five taxonomically different marine bacteria after attachment on two types of glass surfaces, as-received and chemically etched, have been investigated. Contact angle measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray fluorescence spectroscopy (XRF) and X-ray photoelectron spectrometry (XPS) were employed to investigate the impact of nanometer scale surface roughness on bacterial attachment. Chemical modification of glass surfaces resulted in a ～1 nm decrease in the average surface roughness (R _a) and the root-mean-squared roughness (R_q ) and in a ～8 nm decrease in the surface height and the peak-to-peak (R _max) and the 10-point average roughness (R_z ). The study revealed amplified bacterial attachment on the chemically etched, nano-smoother glass surfaces. This was a consistent response, notwithstanding the taxonomic affiliation of the selected bacteria. Enhanced bacterial attachment was accompanied by elevated levels of secreted extracellular polymeric substances (EPS). An expected correlation between cell surface wettability and the density of the bacterial attachment on both types of glass surfaces was also reported, while no correlation could be established between cell surface charge and the bacterial retention pattern.     

Relationship Between Structural Properties and Electrochemical Characteristics of Monolithic Carbon Xerogel‐Based Electrochemical Double‐Layer Electrodes in Aqueous and Organic Electrolytes

The impact of the micropore width, external surface area, and meso‐/macropore size on the charging performance of electrochemical double‐layer capacitor (EDLC) electrodes is systematically investigated. Nonactivated carbon xerogels are used as model electrodes in aqueous and organic electrolytes. Monolithic porous model carbons with different structural parameters are prepared using a resorcinol‐formaldehyde‐based sol–gel process and subsequent pyrolysis of the organic precursors. Electrochemical properties are characterized by utilizing them as EDLC half‐cells operated in aqueous and organic electrolytes, respectively. Experimental data derived for organic electrolytes reveals that the respective ions cannot enter the micropores within the skeleton of the meso‐ and macroporous carbons. Therefore the total capacitance is limited by the external surface formed by the interface between the meso‐/macropores and the microporous carbon particles forming the xerogel skeleton. In contrast, for aqueous electrolytes the total capacitance solely depends on the total surface area, including interfaces at the micropore scale. For both types of electrolytes the charging rate of the electrodes is systematically enhanced when increasing the diameter of the carbon xerogel particles from 10 to 75 nm and the meso‐/macropore size from 10 to 121 nm.     

Development of monoclonal antibody-coated immunomagnetic beads for separation and detection of norovirus (genogroup II) in faecal extract samples

Aims:  The aim of this study is to develop an RT-PCR assay combined with immunomagnetic beads (IMS/RT-PCR) coating monoclonal antibody (Mab) for separation and detection of norovirus (genogroup II) in faecal samples. We furthermore compare its detection limits with IMS/RT-PCR using polyclonal antibody (Pab) and the TRIzol extraction method followed by RT-PCR (TRIzol-RT-PCR).
Methods and Results:  Mab-coated beads and Pab-coated beads were added to a series of tenfold dilutions of faecal extract containing norovirus in 1 ml PBS. After incubation and collection, the RNA was released by heating from virus separated by beads. The tenfold dilutions of faecal were also extracted with TRIzol reagent. The RNA was used as the template for RT-PCR detection (primers: JV12–JV13). IMS/RT-PCR using Mab showed an endpoint in the 10⁻⁷ dilution and was 10² times more sensitive than IMS/RT-PCR using Pab and was at least 10³ times more sensitive than TRIzol-RT-PCR method.
Conclusions:  IMS/RT-PCR using Mab proved to be a more sensitive method of noroviruses (NVs) detection than IMS/RT-PCR using Pab and the TRIzol-RT-PCR method.
Significance and Impact of the Study:  This is the first study to detect NVs with IMS/RT-PCR using Mab, and could serve as a model for future assays when broadly reactive NVs-specific Mabs are developed.     

Se(VI) reduction by continuous-flow reactors packed with Shigella fergusonii strain TB42616 immobilized by Ca2+-alginate gel beads

Selenium at high levels may cause adverse health effects on human beings and endanger aquatic lives due to its toxicity. Se(VI) reduction in continuous-flow reactors packed with Shigella fergusonii strain TB42616 immobilized by Ca²⁺-alginate gel beads was investigated under various hydraulic retention times (HRT) and influent Se(VI) concentrations. Removal efficiency up to 98.8 % was achieved after 96 days operation under an HRT of 5 days and an influent Se(VI) concentration of 400 mg/L. The results showed that the overall selenium removal efficiency was affected by the HRT and the bed height of the reactor but not the influent Se(VI) concentration. The steady-state data were analyzed using a mathematical model and Monod-type kinetics. Biokinetic parameters of half-velocity constants and maximum specific reduction rates were optimized using steady-state data obtained under a range of HRTs (0.73–5.0 days) at a constant influent Se(VI) concentration of 50 mg/L. The model was validated using steady-state data obtained under influent Se(VI) concentrations ranging from 10 to 400 mg/L while maintaining the HRT at 5.0 days. The high correlation coefficients between model calculated Se(VI) and Se(IV) concentrations and the experimental data indicate that the model is robust to predict the performance of the continuous-flow bioreactor.