肠道病毒71型的快速纯化及其鉴定

目的 建立一种简单、快速有效的从细胞培养物中纯化肠道病毒71(EV71)的方法.方法 EV71病毒在恒河猴肾细胞(LLC-MK2)中增殖后,将获得的细胞培养物经反复冻融、聚乙二醇6000沉淀、超速离心、氯化铯垫层超速离心的方法纯化病毒.用聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹(Western blot)和透射电镜(TEM)的方法对纯化病毒进行鉴定,并测定其感染性的滴度及回收率.结果 SDS-PAGE显示出3个条带,相对分子质量分别为3.6×103、3×103和2.6×103与EV71的VP1、VP2和VP3相符合.Western blot证实为EV71特异性条带.经磷钨酸负染后电镜观察,能看到典型病毒颗粒.采用终浓度为10%的聚乙二醇6000沉淀后的病毒的感染性回收率为82.0%,再经氯化铯垫层超速离心后浓缩病毒的感染性回性率为29.0%.结论 聚乙二醇6000沉淀结合氯化铯垫层超速离心的方法比氯化铯密度梯度区带离心方法更简便,易于操作,并且比单独聚乙二醇6000沉淀有更高的纯度,是一种简便、有效的病毒纯化方法.     

序贯消化道净化联合血液灌流治疗急性重度吡虫啉中毒疗效观察

吡虫啉是一种新型的硝基亚甲基类的内吸杀虫剂,具有胃毒和触杀作用。其杀虫机制主要是选择性抑制昆虫神经系统烟碱型乙酰胆碱受体,阻断昆虫神经系统的正常传导,导致昆虫麻痹进而死亡。吡虫啉对人畜均有一定毒害。急性重度吡虫啉中毒病情凶险,死亡率高。彻底清除体内吡虫啉可明显提高重度中毒患者的抢救成功率。序贯消化道净化联合血液灌流治疗急性重度吡虫啉中毒疗效显著。     

竹荪子实体多糖的提取及化学组成

从天然竹荪中经脱脂、采用20、40、60、80℃四个不同温度热水梯度抽提、Sevage法脱蛋白、乙醇沉淀得到四种粗竹荪多糖。对其中在60℃下提取的多糖进行研究。经测定此多糖具有较高的粘度,再对其用丙酮进行分级得到纯度较高的竹荪多糖-3。用气相色谱对其单糖组成结构进行分析,结果表明荪多糖-3是一种以葡萄糖为主的多糖,通过刚果红实验证实此多糖可能具有三螺旋结构。     

Polyhydroxyalkanoates (PHAs) as Biomaterials in Tissue Engineering: Production,Isolation, Characterization

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible biopolymers. These biomaterials have grown in importance in the fields of tissue engineering and tissue reconstruction for structural applications where tissue morphology is critical, such as bone, cartilage, blood vessels, and skin, among others. Furthermore, they can be used to accelerate the regeneration in combination with drugs, as drug delivery systems, thus reducing microbial infections. When cells are cultured under stress conditions, a wide variety of microorganisms produce them as a store of intracellular energy in the form of homo- and copolymers of [R]—hydroxyalkanoic acids, depending on the carbon source used for microorganism growth. This paper gives an overview of PHAs, their biosynthetic pathways, producing microorganisms, cultivation bioprocess, isolation, purification and characterization to obtain biomaterials with medical applications such as tissue engineering.     

Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel

Only L- and N-type high voltage-activated calcium currents (HVA I_Ca) have been demonstrated in identified embryonic spinal motoneurons. However, pharmacological experiments suggest that other HVA I_Ca, including P-type, govern neurotransmitter release at the adult neuromuscular junction. We sought to analyse if embryonic motoneurons express these other I_Ca, using the whole-cell voltage-clamp method on motoneurons purified by a new metrizamide-panning technique from E15 rat embryos. In addition to L-type dihydropyridine-sensitive and N-type ω-GVIA-sensitive currents, motoneurons express two other HVA I_Ca. One has properties related to the P-type channel currents described in Purkinje cells: it is inhibited by the peptide ω-agatoxin-IVA with a maximal effect at 100–200 nM. The inhibited current has a characteristic sustained component during depolarizing test pulses. Furthermore, 50–100 nM concentration of ω-agatoxin-IVA reduce the increase in cytoplasmic calcium concentration observed after depolarization. The other HVA I_Ca is resistant to saturating concentrations of verapamil, ω-conotoxin GVIA and ω-agatoxin-IVA which block L, N and P-type HVAI_Ca, respectively. These results suggest that it is now possible to dissect, using a simple method of purification, the properties of the I_Ca in embryonic mammalian motoneurons and to provide pharmacological evidence for multiple calcium channels which may be involved in regulation of their activity during development.     

高灌蓝越橘多酚氧化酶的提取和纯化

采用丙酮粉沉淀抽滤法、硫酸铵分级沉淀法及苯基琼脂糖疏水性层析法对高灌蓝越橘多酚氧化酶进行提取和纯化。在提取和纯化中，用加入不同的酚吸收剂和抑制剂并比较酶活性的高低来确认最佳方法。结果表明在粗提中加入酚吸收剂、增溶剂和抑制剂提取效果较好，苯基琼脂糖疏水性层析法对此类酶纯化效果较佳并为后来的辅酶的分离提供了较高的纯化倍数。     

A Sustainable Technique to Prepare High-Purity Vanadium Pentoxide via Purification with Low Ammonium Consumption

The general preparation method for V₂O₅ is ammonium salt vanadium precipitation, which inevitably produces large amounts of ammonia nitrogen wastewater. In this paper, we propose an environmentally friendly method for preparing high-purity V₂O₅ with low ammonium consumption. The purity of the V₂O₅ product reaches more than 99% while reducing the level of ammonium consumption. The vanadium precipitation efficiency reaches 99.23% and the V₂O₅ purity of the product reaches 99.05% under the following conditions: precipitation time of 1.5 h, precipitation temperature of 98 °C, initial precipitation pH of 2, ammonium addition coefficient of 2, purification time of 5 min with purification performed twice, purification temperature of 65 °C. In this study, compared with the use of ammonia spirit for vanadium precipitation and ammonium salt vanadium precipitation, the ammonia consumption levels are reduced by 79.80% and 80.00%, and the purity levels are increased by 0.70% and 1.01%, respectively. The compositions of the precipitated (NaV₃O₈∙xH₂O) and purified ((NH₄)₂V₆O₁₆·1.5H₂O) hydrolysis products are characterized via XRD. The TGA results show that NaV₃O₈∙xH₂O contains 1.5 times the amount of crystal water. The FTIR results explain that the two V₃O₈⁻ layers are combined end-to-end to form a V₆O₁₆²⁻ layer. The change of the product image indicates that the purification process includes three stages. Firstly, heating and NH₄⁺ attack expand the V₃O₈⁻ layer. NH4⁺ diffuses more easily into the V₃O₈⁻ layer. Secondly, NH₄⁺ destroys the electrostatic interaction between Na⁺ with the V₃O₈⁻ layer and replacing Na⁺. Finally, V₃O₈⁻ is polymerized into V₆O₁₆²⁻ to keep the crystal structure stable.     

Development of a Novel UPLC-MS/MS Method for the Simultaneous Determination of 16 Mycotoxins in Different Tea Categories

The contamination of potential mycotoxins in tea production and consumption has always been a concern. However, the risk monitoring on multiple mycotoxins remains a challenge by existing methods due to the high cost and complex operation in tea matrices. This research has developed a simple ultra-performance liquid chromatography-tandem mass spectrometry strategy based on our homemade purification column, which can be applied in the detections of mycotoxins in complex tea matrices with high-effectively purifying and removing pigment capacity for 16 mycotoxins. The limits of detection and the limits of quantification were in the ranges of 0.015~15.00 and 0.03~30.00 µg·kg⁻¹ for 16 mycotoxins, respectively. Recoveries from mycotoxin-fortified tea samples (0.13~1200 µg·kg⁻¹) in different tea matrices ranged from 61.27 to 118.46%, with their relative standard deviations below 20%. Moreover, this method has been successfully applied to the analysis and investigation of the levels of 16 mycotoxins in major categories of tea and the monitoring of multiple mycotoxins in processed samples of ripened Pu-erh. In conclusion, the proposed strategy is simple, effective, time-saving, and low-cost for the determination of a large number of tea samples.     

新型无毒防锈颜料三聚磷酸二氢铝研究进展

介绍三聚磷酸二氢铝的性质、合成工艺及应用。采用湿法磷酸净化替代热法磷酸可降低成本,将成为今后此类防锈颜料的发展方向。特别提到昆明理工大学精细化工教研室,开展了肥料级磷酸一铵水溶液添加一定的净化剂,可使磷酸一铵水溶液达到合成三聚磷酸二氢铝的要求。该法称为磷酸一铵净化法,有望成为取代热法磷酸的理想方法。     

Purification of cell culture-derived influenza A virus via continuous anion exchange chromatography on monoliths

The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum-free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM® QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA. The continuous chromatography was implemented as simulated moving bed chromatography (SMB) in a three zone open loop configuration with a detached high salt zone for regeneration.SMBs exploiting 10% and 50% of the monoliths’ dynamic binding capacity, respectively, allowed the depletion of >98% of the DNA and >52% of the total protein. Based on the hemagglutination assay (HA assay), the virus yield was higher at 10% capacity use (89% vs. 45%). Both SMB separations resulted in a ratio of total protein to hemagglutinin antigen (based on single radial diffusion assay, SRID assay) below the required levels for manufacturing of human vaccines (less than 100 µg of protein per virus strain per dose). The level of contaminating DNA was five-times lower for the 10% loading, but still exceeded the required limit for human vaccines. A subsequent Benzonase® treatment step, however, reduced the DNA contamination below 10 ng per dose. Coupled to continuous cultivations for virus propagation, the establishment of integrated processes for fully continuous production of vaccines seems to be feasible.