A Simple Procedure for the Purification and Antiserum Production of Bean Yellow Mosaic Virus

A simple procedure was developed to purify bean yellow mosaic virus from infected faba bean. The procedure included clarification of tissue homogenate by 25% chloroform followed by low-speed centrifugation, virus concentration by polyethylene glycolprecipitation and further purification by agarose-acrylamide gel electrophoresis. The partially purified virus preparation was electrophoresed in 0.5% agarose-2% acrylamide gel for 4 h. Gel bands containing the virus were collected, homogenized, and mixed (1:1) with Freunds adjuvant. Four weekly intramuscular injections and a booster injection four weeks after the fourth injection were given to a rabbit. Antisera collected from the first five bleedings produced high A₄₀₅ readings in ELISA (0.47,5–0.790) with virus-infected faba bean leaves and low readings (0.030–0.065) with healthy tissue. Plates were coated with 5μg/ml of gammaglobulins (IgG) fractionated from the different bleedings of the antiserum prepared and a 1: 1000 dilution ofthe IgG from the third bleeding conjugated to alkaline phosphatase was used.     

A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na⁺ and H⁺ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca²⁺. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.     

Production of Antiserum for Staphylococcal Enterotoxin

Immunization of rabbits with approximately 95% pure enterotoxin B and approximately 20 and 50% pure enterotoxin A yielded specific antisera which required no and little absorption, respectively, when used in the slide double-diffusion test. Methods for purifying enterotoxin A and for the production and absorption of antisera are presented.     

Purity Control of the Production of Baker''s Yeast Employing a Fluorescent Antiserum

A simple staining procedure for the rapid detection of wild yeasts contaminating baker's yeast during the course of industrial production is described. Fluorescein-labeled, specific antiserum against Saccharomyces cerevisiae is applied to smears of baker's yeast which are then examined by ultraviolet microscopy. Optimal results are obtained with the combined phase contrast and fluorescence which makes the S. cerevisiae appear green, whereas cells of wild yeasts are visible in bright red counterstain. With this method, wild yeasts could be identified in baker's yeast at a dilution of 1:10,000.     

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

BackgroundEnvenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.ConclusionHere we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.     

A Novel Polystyrene Dish for the Production of Anaerobiosis

S ummary : The design and construction of a polystyrene dish to be used with alkaline pyrogallol for the production of anaerobic conditions is described. Paper mats are used to carry the reducing system, and the use of cellulose adhesive tape to effect an airtight seal is recommended.     

介绍一种固定蛇类标本外翻半阴茎的制作方法

蛇类半阴茎的形态特征在蛇类分类、进化和系统发育等研究中具有重要价值.本文介绍了一种改进的固定蛇类标本外翻半阴茎的制作方法.     

A Simple Set of Plasmids for the Production of Transgenic Plants

A new set of plasmids for plant transgenic studies was developed. Its strong point is that independent gene cassettes are connected within one binary vector by the restriction endonuclease-based technique only. Using the set, two overexpressing cassettes and three RNA interference (RNAi) cassettes were successfully introduced into rice. Our plasmid set is useful for producing commercial transgenic plants, especially in the case of rice.     

A New Fixation Procedure for Study of the Histaminergic Neurons by Immunoelectron Microscopy using the Direct Antiserum against Histamine

A new perfusion protocol was developed to detect histaminc-like immunoreactive neurons at the electron microscopic level. By stepwise perfusion of 1-ethyl-3(3-diamethylaminopropyl)-carbodiimide and paraformaldchyde solutions, the brain block could be cut with a vibratome and the immunoreactivity could be detected using the avidin-biotin-peroxidase-complex method. We used this method to study the ultrastructure and synaptic relations of the histaminergic neurons in the postmanunillary caudal magnocellular nucleus of the rat hypothalamus. This method should also be useful for examination of histaminergic neurons in other tissues and the synaptic relations of histaminergic neurons with other neurotnunsmitter-containing neurons by double immunostaining.     

A Novel Undulatory Propulsion Strategy for Underwater Robots

Stingrays can undulate their wide pectoral fins to thrust themselves and swim freely underwater.Many researchers have used bionics to directly imitate their und...     

Production and Characterization of a New Specific Antiserum Against the Taurine Putative Biosynthetic Enzyme Cysteine Sulfinate Decarboxylase

Abstract: We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were im munostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.     

大规模合成长链RNA的简易低成本工艺

由于现有技术所限,RNA分子长度和二级结构往往成为RNA合成困难的主要原因.提供了一种简单低成本大规模制备和纯化长链RNA药物的新工艺,尤其针对具有稳定二级结构的长链RNA药物.采用引物延伸方法替代PCR和线性质粒DNA方法制备线性DNA模板可减少步骤及降低污染,然后用T7 RNA聚合酶转录制备的甲氧基修饰的线性DNA模板获得高均一度的长链RNA,转录粗产物直接用source 15Q阴离子HPLC分离T7 RNA聚合酶、rNTP、转录中断产物、内毒素和模板DNA等,从而获得高纯度RNA终产物.该工艺无需繁琐的酚/氯仿抽提和RNA变性,尤其适用于RNA的大量制备.     

Antifungal Susceptibility, Exoenzyme Production and Cytotoxicity of Novel Oximes against Candida

Novel oximes were synthesized, their in vitro antifungal activity against Candida was evaluated and their cytotoxicity was determined. The procedure used for the synthesis of the oximes is aligned with the current green chemistry trend; water is employed as the solvent in this reaction. The minimum inhibitory and minimum fungicidal concentrations of the oximes were evaluated using the CLSI M27-A3 method. The influence of these compounds on the inhibition of the production of hydrolytic enzymes, phospholipase and proteinase by Candida was also investigated. The compounds showed a good ability to inhibit phospholipase, with a 50 % reduction in most cases. However, the tested compounds did not affect proteinase. The current results showed a substantial reduction in the phospholipase production, which suggests that compounds of this class may interfere with host infection and disease progression. The oximes examined showed lower fungicidal activities than fluconazole but interfered significantly with the expression of phospholipase. Some of the oximes included in this study could be a suitable matrix for the development of novel antifungal compounds.     

长爪沙鼠IgG的纯化及抗血清的制备

目的 纯化长爪沙鼠血清IgG,制备兔抗长爪沙鼠IgG抗血清。方法 采用Hitrap Protein G亲和层析预装柱来纯化长爪沙鼠血清IgG;通过SDS-PAGE电泳和Western-Blotting免疫印迹法对长爪沙鼠血清IgG进行纯度鉴定,免疫兔子制备抗血清。结果 7 mL长爪沙鼠血清纯化得到11 mg IgG;电泳和免疫印迹测定,IgG纯度大于95%;用纯化的IgG作抗原制备了兔抗血清,免疫双扩散测定效价达1∶32。结论 建立了长爪沙鼠血清IgG的纯化方法,制备了长爪沙鼠IgG抗血清,证实长爪沙鼠血清IgG和Protein G具有较高的亲和性。     

硫酸软骨素荧光传感器的构建新策略

硫酸软骨素（chondroitin sulfate,CS）是一类从动物组织中提取的蛋白类黏性多聚糖,在人体免疫调节、缓解关节疼痛、抗凝活性和保护神经元等方面发挥效用,开发硫酸软骨素传感器对生物医药行业研发相关药剂、保健产品质量的粗筛具有重要意义。利用荧光传感器制备简便、响应迅速、选择性强的特点,用阳离子聚合物PDDA诱导芘基分子HPTS自组装,导致荧光淬灭构建传感器。研究结果显示,待测物的加入引起510 nm处荧光强度的恢复,最终建立了实际样品中CS的快速分析方法。构建的硫酸软骨素荧光传感器在浓度0～5 μmol·L-1间呈现良好的线性关系,线性方程为y=1.907 52+0.273 59x(R2=0.998 2),检出限为2.8 μmol·L-1,样品加标回收率在104%～121%之间。该传感器具有水溶性好、特异性强、直观可视等特点。     

Production and characterization of a generic antibody against group A trichothecenes

An antibody against group A trichothecenes was produced after immunization of rabbits with an immunogen prepared by conjugation of T-2 toxin to bovine albumin at the C-8 position. T-2 toxin was first converted to 3-acetylneosolaniol (3-Ac-NEOS) and then to its hemisuccinate (HS) before conjugation to the protein. The rabbits showed a quick immune response after immunization of the new conjugate. The antibody produced bound with tritiated T-2 toxin, T-2 tetraol tetraacetate, and diacetoxyscirpenol (DAS) and showed good cross-reactivities with most of the group A trichothecenes. The concentrations causing 50% inhibition of binding of 3H-T-2 toxin to the new antibody by unlabeled T-2, acetyl-T-2, 3'-OH-T-2, DAS, 3-Ac-NEOS-HS, 3'-OH-Ac-T-2, T-2 tetraol tetraacetate, iso-T-2, 3-Ac-NEOS, Ac-DAS, and 3,4,15-triacetyl-7-deoxynivalenol were found to be 0.34, 0.34, 0.6, 2.5, 4, 10, 18, 24, 100, 200, and 300 ng/assay, respectively; for HT-2, T-2 triol, and T-2 tetraol, the concentration was greater than 1000 ng/assay. Nivalenol, deoxynivalenol (DON), 15-acetyl-DON, and triacetyl-DON, did not inhibit the binding at 1000 ng/assay. The practical application of using this new antibody for radioimmunoassay (RIA) of trichothecene was tested by spiking T-2 toxin to corn. T-2 toxin was then extracted with acetone, subjected to a simple Sep-Pak C-18 reversed-phase treatment, and analyzed by RIA. The overall recovery for 18 samples spiked with 10 to 50 ppb of T-2 toxin was 94.22%.