研究报告：鳗弧菌（Vibrio anguillarum）核酸适配体的筛选及其结合蛋白的分离鉴定

目的鳗弧菌（Vibrio anguillarum）是水产养殖中的重要条件致病菌,每年给水产养殖业造成巨大的经济损失,研究其致病机制、对其进行快速的检测鉴定是其病害防治的前提和基础。核酸适配体因其高亲和力、高特异性等多种优点,在微生物的靶标分析、检测鉴定以及致病机制等多个领域都呈现出较好的应用潜力。因此,筛选鳗弧菌的核酸适配体,利用核酸适配体对鳗弧菌相关位点进行分析鉴定,不仅能为鳗弧菌的检测鉴定提供一个新的手段,对于探索鳗弧菌相关位点在其病害防治中的作用也具有重要意义。方法以鳗弧菌为靶目标,采用每轮测序的SELEX筛选方法,从高频序列中筛选鳗弧菌的核酸适配体;采用单链DNA浓度法测定核酸适配体的亲和力,研究核酸适配体对鳗弧菌的亲和特异性;采用Origin软件、选择反比例函数（Hyperbola函数）进行非线性拟合,获得核酸适配体的亲和常数（K_d）和最大亲和力（A_m）;采用磁分离技术和聚丙烯酰胺凝胶电泳分离纯化出核酸适配体H5的结合蛋白,通过质谱对该蛋白质进行分析鉴定,并利用Prabi、Phyre2、Psortb 3.0等在线网站分析该结合蛋白的...     

研究报告: 基于核酸适配体的胶体金比色法检测鳗弧菌

目的 鳗弧菌（Vibrio anguillarum）可引起鲑鱼、鳗鲡、鲈鱼和牙鲆等多种水产养殖动物的疾病，是水产养殖中的一种重要病原菌，对其进行快速检测是确保水产养殖安全和食品安全所必需的。方法 本文利用鳗弧菌与其核酸适配体之间较强的亲和力，通过鳗弧菌夺取胶体金颗粒表面的核酸适配体，使胶体金溶液的吸光度发生变化，从而建立一种可定量检测鳗弧菌的方法。结果 该方法对鳗弧菌的吸光度值显著高于对溶藻弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌和迟钝爱德华氏菌等非目标菌的吸光度值（P<0.01），并在1~10⁵ CFU/ml的检测范围内呈现较好的线性关系。用该方法对不同盐度和鱼体组织样品进行加标回收检测，结果显示回收率和相对标准偏差等指标都符合相应检测标准。结论 该检测方法对鳗弧菌有较好的特异性，可用于水产品或食品中鳗弧菌的定量检测。     

环境因子对鳗弧菌生物膜形成的影响

【背景】鳗弧菌是海产动物弧菌病的主要病原,在海水水域中广泛存在。鳗弧菌为了适应环境变化会生成生物膜,形成自我保护,对其防治是水产养殖行业的一大难题。【目的】探讨致病性鳗弧菌(Vibrio anguillarum)BYK0638生物膜的形成特性,为进一步研究鳗弧菌生物膜形成机制和致病机理提供参考。【方法】采用改良的微孔板法研究静置培养条件下鳗弧菌(V.anguillarum)BYK0638在96孔酶标板上的成膜情况,CCK-8法(Cell counting kit-8)定量检测生物膜中鳗弧菌的活力。【结果】鳗弧菌BYK0638能够在聚苯乙烯酶标板上形成稳定而明显的生物膜,其生物膜的OD450值在24 h达到峰值,60 h后趋于稳定;在107-108 CFU/m L范围内,鳗弧菌生物膜的OD450值显著高于其他试验组(P0.05);25°C时的生物膜OD450值显著高于其他温度生物膜的形成量;在p H 4.0-11.0范围内,当p H值为7.0时鳗弧菌形成的生物膜量最高,在p H值为3.0和12.0时鳗弧菌几乎不形成生物膜;在TSB培养基中加入0.03-2.00 mmol/L Ca Cl2,鳗弧菌生物膜形成量与未添加Ca Cl2对照组无显著性差异;在TSB培养基中加入0.03 mmol/L Mg Cl2,可促进鳗弧菌生物膜形成;Na Cl浓度为5%时,形成的生物膜OD450值最高;鳗弧菌在大黄鱼表皮黏液、肝脏、前肠、后肠组织提取液包被的96孔酶标板上形成的生物膜显著高于其他黏液和组织提取液包被组(P0.05)。【结论】致病性鳗弧菌BYK0638能形成稳定而明显的生物膜,其生物膜形成与外界环境因子变化有密切的关系,培养时间、初始菌浓度、温度、p H、Mg2+、盐度及不同组织和黏液等各种环境因子均能显著影响鳗弧菌生物膜的形成。     

鳗弧菌毒力相关基因的研究进展

鳗弧菌是引起多种海水鱼类出血性败血症的病原菌。其致病机理与各个毒力基因的协同作用密切相关。文中综述了鳗弧菌的主要毒力基因,包括编码外毒素、粘附因子、侵袭因子、细胞表面成分以及铁吸收系统的基因和部分检测方法。     

鳗弧菌毒力相关基因的研究进展

鳗弧菌是引起多种海水鱼类出血性败血症的病原菌。其致病机理与各个毒力基因的协同作用密切相关。文中综述了鳗弧菌的主要毒力基因,包括编码外毒素、粘附因子、侵袭因子、细胞表面成分以及铁吸收系统的基因和部分检测方法。     

溴化呋喃酮对鳗弧菌群体感应调控行为的抑制研究

以群体感应抑制剂—溴化呋喃酮为研究对象,探究其对鳗弧菌群体感应相关基因和致病因子的抑制作用。利用荧光定量PCR检测亚抑菌浓度溴化呋喃酮对鳗弧菌van I/R和luxS基因表达量的影响,利用鞭毛染色观察溴化呋喃酮对鳗弧菌生物膜以及鞭毛形成的作用,通过平板扩散法检测溴化呋喃酮对鳗弧菌胞外酶的作用。结果表明,溴化呋喃酮能够显著抑制鳗弧菌van I和luxS基因的表达量,溴化呋喃酮能够降低鳗弧菌分泌蛋白酶和明胶酶能力,能抑制鞭毛生长和生物膜的形成并降低其运动能力。     

六种弧菌多重PCR快速检测方法的建立

目的:检测鳗弧菌、哈氏孤菌、副溶血孤菌、溶藻胶弧菌、霍乱弧菌和创伤孤菌六种水产常见病原菌.方法:以toxR-toxS为靶基因设计引物,建立了一种多重PCR( multiplex PCR,mPCR)快速检测方法.结果:本研究设计的mPCR引物特异性强,与其他弧菌及非孤菌无交叉反应,每次反应的敏感性为10-100 CFU(cell forming unit)/每个反应.结论:应用建立的mPCR方法结果稳定可靠,有望成为检测病害弧菌的有效工具.     

一株牙鲆皮肤溃烂症病原菌的鉴定

从山东荣成养鱼场发病牙鲆分离到一株病原菌M3,革兰氏阴性,杆状,能运动,菌落半透明,用BIOLOG细菌鉴定系统不能鉴定。通过16S rDNA序列分析和同源性检索发现M3菌株与弧菌属的同源性较高,为94%～98%。系统发育学分析表明菌株M3与鳗弧菌关系最近,相似性为996%,其生化性状也和鳗弧菌的特征相似,故可把M3定为鳗弧菌(Vibrio anguillarum)。     

六种弧菌多重PCR快速检测方法的建立

目的:检测鳗弧菌、哈氏弧菌、副溶血弧菌、溶藻胶弧菌、霍乱弧菌和创伤弧菌六种水产常见病原菌。方法:以toxR-toxS为靶基因设计引物,建立了一种多重PCR(multiplex PCR,mPCR)快速检测方法。结果:本研究设计的mPCR引物特异性强,与其他弧菌及非弧菌无交叉反应,每次反应的敏感性为10-100 CFU(cell forming unit)/每个反应。结论:应用建立的mPCR方法结果稳定可靠,有望成为检测病害弧菌的有效工具。     

哈维氏弧菌适配子的SELEX筛选及其亲和特异性研究

哈维氏弧菌是水产养殖中的重要条件致病菌,对其进行快速、准确地检测和鉴定是相关病害防治的基础和关键.适配子具有亲和力高、特异性强、稳定性好等优点,在微生物的检测和鉴定方面呈现出广泛的应用前景.本研究以哈维氏弧菌为靶目标,采用SELEX技术,即指数级富集配体的系统进化技术,筛选其特异性适配子.经15轮筛选后,随机ssDNA文库的亲和力从3.51上升到58.95,提高了15.8倍.筛选出的适配子富集库经克隆、测序后得到52条不同序列,根据同源性将这些序列分成8个家族,其中第1和第2家族的适配子数量最多,超过总数的50%.通过深入分析,筛选出6个对哈维氏弧菌有显著亲和特异性(P0.01)的高频适配子,其中5个高频适配子(S1、S25、S26、S27、S35)对哈维氏弧菌有较高的亲和力,相应的亲和常数Kd值分别为(32.6±7.1)、(45.3±10.1)、(24.7±5.8)、(34.8±5.6)、(12.9±4.0)nmol/L.本文还对高频适配子的产生机制及其应用价值进行了探讨.本文首次筛选出了对哈维氏弧菌具有较高亲和特异性的适配子,为后续利用适配子进行哈维氏弧菌的检测和鉴定奠定了基础.     

Functional RNA microarrays for high-throughput screening of antiprotein aptamers

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.     

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr‐activated sepharose‐4B affinity chromatography

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA‐aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (k_d = 2.3 × 10^?11). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.     

Screening of DNA Aptamer Against Mouse Prion Protein by Competitive Selection

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody. Aptamers are a novel type of ligand composed of nucleic acids. DNA aptamers in particular have many advantages over antibodies. Therefore, we tried to isolate the DNA aptamer for mouse PrP. We developed a competitive selection method and tried to screen the DNA aptamer with it. In the fourth round of selection, several clones of the aptamer with an affinity to PrP were enriched, and clone 4-9 showed the highest affinity of all. The investigation by aptamer blotting and Western blotting showed that clone 4-9 was specifically able to recognize both α-PrP and β-PrP. Moreover, it was indicated that clone 4-9 could recognize the flexible region of the N-terminal domain of PrP. These characteristics suggest that clone 4-9 might be a useful tool in prion-disease diagnosis and research.     

肝脏去唾液酸糖蛋白受体特异性RNA适配子的SELEX筛选与鉴定

目的获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体（asialoglycoprotein receptor,ASGPR）的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础。方法合成一个长度为115nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,以肝脏ASGPR大亚基为靶蛋白,采用SELEX（systematic evolution of ligands by exponential enrichment）技术筛选具有高亲和力的AsGPR特异性RNA适配子;通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力。结果经过12轮筛选获得了具有高亲和力的肝脏ASGPR特异性RNA适配子。结论成功地筛选出了具有离亲和力的肝脏ASGPR特异性RNA适配子库。     

Assays for cytokines using aptamers

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.     

Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin,NTS-1

G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy. Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography. Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers. Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants. Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays. Eight aptamers demonstrated specificity for the neurotensin receptor. One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity. P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells. P19 represents the first example of a GPCR-specific RNA aptamer.     

Selection of RNA aptamers against human influenza virus hemagglutinin using surface plasmon resonance

Aptamers are functional nucleic acids possessing high affinity and specificity to their cognate ligands and are isolated from a library of nucleic acids by iterative rounds of selection and amplification. In the current study, we used surface plasmon resonance (Biacore) as an efficient methodology for selecting aptamers that bind to hemagglutinin (HA) of human influenza virus. This procedure allowed us to monitor and select the target-bound aptamers specifically and simultaneously. These studies not only yielded an aptamer that binds to the HA of influenza virus with high affinity but also revealed the consensus sequence, 5'-GUCGNCNU(N)(2-3)GUA-3, for HA recognition.