金属螯合亲和层析的应用研究进展

金属螯合亲和层析是近30年来出现的一种新型的分离纯化技术,它以配基简单、吸附量大、分离条件温和、通用性强等特点,逐渐成为分离纯化蛋白质等生物工程产品最有效的技术之一.其在蛋白质、酶和核苷酸纯化以及蛋白质分析等方面具有广泛的应用.而随着研究的进一步深入,其新应用也越来越广泛的被发现.     

Affinity Purifications of Aldose Reductase and Xylitol Dehydrogenase from the Xylose-Fermenting Yeast Pachysolen tannophilus

Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.     

基于功能化磁珠快速提取植物病毒RNA方法的建立与优化

植物病毒病是制约作物生产的主要病害之一。及时明确其病毒病原和发展规律是有效控制其大规模传播的前提。而现有植物病毒病检测技术存在周期长、步骤繁琐、检测环境严苛等缺点。本研究以烟草花叶病毒 (Tobacco mosaic virus,TMV)为模型,基于碱基互补配对原则设计针对TMV的功能化磁珠（CMBs-ACPTMV）进行RNA提取,并对功能化磁珠的制备条件、提取反应条件以及灵敏性和稳定性等性能进行优化分析。结果表明,当添加4 μmol捕获探针（ACPTMV）、0.08 mg羧基磁珠（CMBs）时,所制备的CMBs-ACPTMV吸附RNA的能力最好;当提取时间为3 min时,CMBs-ACPTMV提取RNA的效果最好,而改变CMBs-ACPTMV的提取温度时其提取能力无明显变化;性能评价分析发现,CMBs-ACPTMV的灵敏度可达2.5 ng/μL,且检测稳定性较好。与常规RNA提取技术相比,CMBs-ACPTMV在检测时间和样品消耗量上具有突出优势。本研究所建立的功能化磁珠提取法快速、安全和简便,只需简易设备便可实现植物病毒RNA的快速提取,具有广阔的应用前景。     

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies¹. Since poliovirus is sensitive to conformational conversion² when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch^3,4 is the micro protein A-immunoprecipitation test⁵. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies^6,7. However, as another opportunity, these interesting and stable single-domain antibodies⁸ can be easily engineered with different tags. The widely used (His)₆-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with ³⁵S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.     

高亲和力莱克多巴胺单克隆抗体的研制及ciELISA检测方法的建立

  用混合酸酐法（MA）将莱克多巴胺（RAC）偶联于牛血清白蛋白（BSA）,合成人工抗原BSA-RAC,用UV和SDS-PAGE鉴定;用BSARAC免疫Balb/c小鼠,细胞融合技术建立高亲和力RAC单克隆抗体（mAb）杂交瘤细胞株,体内诱生腹水法制备RAC mAb;应用RAC mAb研制RAC残留快速检测ciELISA试剂盒（RAC-Kit）,并测定其性能。结果表明,BSA-RAC偶联成功,分子结合比为24.5∶1;筛选出3株杂交瘤细胞,其中最好的4D8株亲和常数（Ka）为1.65×1010L/mol; RAC-Kit的检测限为0.5ng/ml,检测范围为0.5～151ng/ml,饲料样、猪尿样的平均添加回收率为87.2%,89.4%,平均批内和批间变异系数小于15%,与多巴酚丁胺的交叉反应率（CR%）为9.7%,与其它化合物无CR,RAC-Kit在4 ℃保存期为180d。     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

高亲和力克伦特罗单克隆抗体的研制及免疫试纸检测方法的建立

重氮化法将克伦特罗（CL）偶联于BSA合成免疫抗原BSA-CL,戊二醛法（GA）合成包被抗原OVA-CL,用BSA-CL免疫Balb／C小鼠。细胞融合技术建立高亲和力CL单克隆抗体（CL mAb）杂交瘤细胞株,体内诱生腹水法制备CL mAb,并测定其亲和性。依据胶体金免疫层析试验（GICA）原理,应用CL mAb研制CL残留快速检测免疫试纸（CL-Srip）,并测定其性能。结果表明,筛选出16株杂交瘤细胞,其中亲和力最高的为3C12—6H6,亲和常数（Ka）为1．76×10^10L／mol。CL-Strip的标准曲线呈典型的S型,符合4参数logit曲线拟合,目测检测限为0．5μg／L;其敏感性与竞争ELISA试剂盒和GC／MS相当,符合率为100％。CL-Strip具有具有敏感、特异、快速、简便的特点,可推广应用于CL残留的快速检测。     

Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N₃), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.     

Efficient Isolation of Pure and Functional Mitochondria from Mouse Tissues Using Automated Tissue Disruption and Enrichment with Anti-TOM22 Magnetic Beads

To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.     

Development of a Rapid and Efficient Method for Non-Lethal DNA Sampling and Genotyping in Scallops

Non-lethal DNA sampling has long appealed to researchers studying population and conservation genetics, as it does not necessitate removing individuals permanently from their natural environment or destroying valuable samples. However, such an approach has not yet been well established in bivalves. In this study, we demonstrate that the gill represents a good source of tissue for non-lethal sampling in scallops. Removal of a few gill filaments caused no noticeable behavioral abnormalities or increased mortality rates in Zhikong scallop (Chlamys farreri) during a three-month period of observation. To facilitate rapid gill-based DNA extraction, six methods (MA-MF) were designed and evaluated, each requiring less than one hour of processing time. The optimal method was identified as MF, in terms of maintaining DNA integrity and genotyping accuracy. Further optimization of MF method by orthogonal experimental design suggested that the utilization of gills could be limited to 2 mg of sample, which is sufficient for performing up to 20,000 PCR reactions. We also demonstrate the excellent cross-species utility of MF in two additional scallop species, Yesso scallop (Patinopecten yessoensis) and bay scallop (Argopecten irradians). Taken together, our study provides a rapid and efficient approach for applying non-lethal DNA sampling in bivalve species, which would serve as a valuable tool for maintaining bivalve populations and conservation genetics, as well as in breeding studies.     

An Affinity Chromatographic Reactor for Highly Efficient Turnover of Dissociable Cofactors

A conjugated enzyme system of alcool dehydrogenase and lactate dehydrogenase was immobilized in an ultrafiltration hollow fiber tube, which was inserted in a fine nylon tube to form a hollow-fiber-capillary reactor. To this reactor, the substrates, pyruvate and ethanol, were supplied continuously. The necessary cofactor, NAD, was supplied as a pulse for a short time. The retention time of NAD in the reactor, estimated from the response curve of lactate produced, was much longer than those of the other substrates and products because of the strong adsorption of NAD to the immobilized enzymes through affinity. Therefore, the reactor could produce lactate from pyruvate for a long time without any more NAD. As a typical case, when the enzyme concentration is sufficiently high, the estimated retention time of NAD was 50 times as long as those of other materials so that the NAD turnover obtained was 412,000. The effects of NAD pulse concentration and the immobilized enzyme concentration on the retention time of NAD and NAD turnover were investigated experimentally and theoretically.     

一种快速、高效花生植株再生体系的建立

快速、高效、重复性好的植株再生体系是转基因育种的基础;本研究以14份不同花生品种的胚小叶为外植体,利用不同激素浓度、组合和不同花生基因型筛选最佳芽诱导培养基、伸长培养基和高效再生基因型。结果表明最佳丛生芽诱导培养基为MSB;＋0．2mg·L-1NAA＋6mg·L-1 6-BA,诱导率为89．50％;最佳伸长培养基为MSB5＋0．2mg.L-1 NAA＋3mg’L-1 6-BA和MSB;＋O．2mg·L～NAA＋4mg·L-1 6-BA＋2mg·L～GA,交替培养,每个丛生芽伸长数达到7．24,时间缩短至3-4周。不同品种再生率的变幅在25．51％～93．01％,大于80％的品种有‘麻油1-1’、‘弗落蔓生’、‘濮花23号’、‘海花1号’。利用‘弗落蔓生’在15周内得到了生根组培苗。     

快速高效的DNA连接

DNA片段的连接在基因操作过程中应用广泛。通常情况下利用T4DNALigase必须经过长达16小时才能完成连接反应,而且反应常常受到DNA末端结构等的影响。宝生物工程（大连）有限公司（TaKaRa）的DNA连接系统（TaKaRaDNALigationKitVer。2）由于其独特的反应液体系,可以将反应时间缩短至半个小时,且不受DNA结构等的影响,大大地节省了反应时间,方便了实验操作。此外,在使用本试剂盒时,由于只需简单地向DNA溶液中加入一种溶液,可以在极小的反应体积下,短时间内高效地完成各种形式的DNA连接（图1、2）。并且反应液可以直接用…     

文蛤微卫星DNA的筛选及其特性分析

采用磁珠富集分离法从文蛤(Meretrix meretrix)的基因组中筛选得到49条微卫星DNA序列,其中两碱基重复有3种类型36个序列,四碱基重复有14种类型26个序列.重复次数在5～30之间的序列占75.6%,30次以上重复的序列占24.4%,最高重复次数为100.根据重复单元的排列特点,完美型、非完美型及混合型序列所占比例分别为61.2%、14.5%和24.5%.本研究中构建的文蛤微卫星文库将在文蛤种质资源评价及分子遗传学研究中发挥作用.     

A Criterion for the Complete Deposition of Magnetic Beads on the Walls of Microchannels

This paper analyzes numerical simulations of the trajectories of magnetic beads in a microchannel, with a nearby permanent cubical magnet, under different flow and magnetic conditions. Analytically derived local fluid velocities and local magnetic forces have been used to track the particles. A centered position and a lateral position of the magnet above the microchannel are considered. The computed fractions of deposited particles on the walls are compared successfully with a new theoretically derived criterion that imposes a relation between the sizes of the magnet and the microchannel and the particle Stokes and Alfvén numbers to obtain the complete deposition of the flowing particles on the wall. In the cases in which all the particles, initially distributed uniformly across the section of the microchannel, are deposited on the walls, the simulations predict the accumulation of the major part of particles on the wall closest to the magnet and near the first half of the streamwise length of the magnet.     

万古霉素的磁性亲和吸附分离

磁性亲和分离技术是近年新兴的一个分离方法,它可直接从初始样品液体中(无论是浑浊或清亮液)分离目标产物,克服了传统色谱方法需要离心和过滤除杂质的步骤,分离过程所用仪器极其简单,大大降低了操作费用,且易于实现规模化;此外,同亲和色谱一样也具有高的特异性及应用范围广等优点.使其在蛋白质、细胞的分离方面表现出了巨大的应用前景[1-4].万古霉素是一个多肽类的抗菌素,临床上用于治疗耐甲氧苯青霉素葡萄糖球菌的感染.目前虽有多种纯化方法[5,],但大多工艺复杂,回收率较低,尤其是在纯化过程中,万古霉素极易降解,迫使人们寻求更好的纯化方法来解决这个问题.     

纳米磁性颗粒标记的免疫层析法定量检测人乙型肝炎表面抗原

目的:应用纳米磁性颗粒标记的免疫层析法,研制可应用于乙肝表面抗原(HBsAg)快速定量检测的层析试纸条。方法:用1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)/N-羟基琥珀酰亚胺(NHS)交联的方法标记纳米磁珠,喷膜仪喷点硝酸纤维膜;根据双抗体夹心法原理建立免疫层析试纸条,对HBsAg特异性抗体捕获的磁信号进行检测,并对磁信号检测结果进行统计学分析和评价。结果:建立了HBsAg纳米磁性免疫层析试纸条,最低限度检测为0.1 ng/mL的HBsAg抗原,检测灵敏度达到了同类产品ELISA分析法的标准,且检测时间控制在5 min内;经检测临床血清标本证实,该方法可根据磁信号定量检测乙肝患者血清中HBsAg的浓度。结论:HBsAg纳米磁性免疫层析方法具有简单快速、灵敏度高的特点,可应用于临床血清样本中HBsAg的检测;该方法为体内极微量抗原抗体的快速检测建立了新模式。