基于核酸适配体的荧光分析法定量检测MUC1粘蛋白

MUC1粘蛋白是一种高糖化、高分子量的糖蛋白,其在乳腺癌细胞中高度异常表达,其特异性高于组织多肽抗原,敏感性高于癌胚抗原,因此MUC1在乳腺癌诊断中具有很高的临床应用价值,而建立高灵敏的MUC1蛋白定量检测方法对临床诊断具有重要的意义.该研究建立了基于核酸适配体-滚环扩增（RCA）和氧化石墨烯-荧光共振能量转移（GO-FRET）技术的MUC1黏蛋白定量检测技术,实现了MUC1粘蛋白准确、灵敏的定量检测.结果表明,该方法定量检测线性范围为50~1 000 pg/mL,检测限为28.05 pg/mL,定量限为45.57 pg/mL,在人血样品中的回收率为96%~104%.     

利用SELEX技术筛选小檗碱核酸适配体及其亲和力表征

小檗碱是一种价格低廉且生物安全性良好的小分子化合物,筛选能与小檗碱互相作用的核酸片段,旨在开发新型遗传元件并拓展其在生物技术领域的应用。利用指数富集的配基系统进化技术（SELEX）,经8轮实验从随机单链DNA文库中筛选出4条能与小檗碱高度特异性结合的核酸适配体。对其中两条重现性较好的适配体进行结构模拟分析表明,适配体序列富含鸟嘌呤和胞嘧啶且多形成茎环及G-四链体结构。亲和力测定结果显示,其中的2条核酸适配体与小檗碱作用的解离常数K_d都处于nmol级,表现出较高的亲和力,适配体S1的K_d值最小,仅为523 nmol/L,与小檗碱亲和力最大。     

水生生物病原核酸适配体研究进展

核酸适配体(Aptamer)是指利用指数富集配体系统进化(Systematic evolution of ligands by exponential enrichment,SELEX)技术筛选得到的能特异性识别和结合靶标的ssDNA或RNA分子,具有高亲和力和高特异性的特点,在快速检测和靶向治疗方面应用广阔。近年来,水生生物细菌和病毒性病原疾病的频繁暴发,严重制约了水生态的健康以及水产养殖业的迅速发展。核酸适配体作为一种新型的识别分子,在水生生物病原的应用上也已取得了一些进展。本文对水生生物细菌和病毒性病原核酸适配体的筛选,及其在检测和治疗领域的研究现状进行综述,并对该领域核酸适配体技术的应用方向进行展望。     

Novel protein detection method based on proximity-dependent polymerase reaction and aptamers

In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary sequence at 3′-end were designed for the two distinct epitopes of the protein. Association of the two aptamers with thrombin resulted in stable hybrids due to the proximity of 3′-end, then polymerase reaction was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green I. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9 pmol/L.     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

适配体修饰的纳米金比色法检测牛奶中的三聚氰胺

以三聚氰胺作为检测目标,设计合成了能与三聚氰胺特异性结合的适配体,选取表面活性剂邻苯二甲酸二乙二醇二丙烯酸酯(PDDA),将纳米金比色法与适配体的分子识别相结合,确立了一种简单、高效、快速检测三聚氰胺的新方法.在三甲基硅丙磺酸(pH 7.0)缓冲溶液条件下,此反应体系的最佳条件为25nmol/L适配体,0.44nmol/L PDDA,反应时间为20min,通过可见光吸收光谱可检测的检测下限为34nmol/L.在牛奶样品的检测中,三聚氰胺的加标回收率为90%~120%,可以满足牛奶样品中三聚氰胺残留的精确检测要求.     

结合适配体技术的磁性粒子表面蛋白分子印迹材料研制

本文将具有高度特异性的生物分子核酸适配体引入分子印迹技术,并结合磁性纳米粒子材料比表面积大、操作简便性和易于修饰性于一体,制备出了新型蛋白分子印迹材料.首先获得性能更好的硅烷化的磁性微球,之后加入功能单体、交联剂和衍生化适配体,以溶菌酶为模板分子,制备出磁性溶菌酶蛋白分子印迹适配体-纳米粒子.实验结果显示,不同材料的蛋白吸附能力顺序由高到低分别为结合适配体的蛋白印迹材料,结合适配体的非蛋白印记材料,蛋白印迹材料和无适配体的非蛋白分子印记材料.初步研究表明,适配体与磁性粒子的结合可以有效提高蛋白质分子印迹材料的印迹效果.     

特异性检测卵形鲳鲹源哈维氏弧菌的核酸适配体的筛选与鉴定

哈维氏弧菌(Vibrio harveyi)是海水养殖动物常见的主要致病菌之一,严重危害着我国华南沿海地区水产养殖业的健康可持续发展。针对哈维氏弧菌的快速检测诊断技术和抑菌技术等方面进行系统研究,并开发具有市场发展潜力的快速检测试剂盒和抗菌功能产品,对控制哈维氏弧菌的危害极其重要。为此,本研究以哈维氏弧菌为靶标,利用指数富集的配基系统进化(Systematic Evolution of Ligands by Exponential Enrichment, SELEX)技术,筛选特异性识别哈维氏弧菌的核酸适配体,并对核酸适配体的性质进行系统性研究。结果显示,核酸适配体H10、H13能高特异性和高亲和性地识别哈维氏弧菌,并且无毒副作用。同时,靶标性质分析表明核酸适配体(H10,H13)的靶标可能是细胞膜蛋白或膜蛋白相关成分。本研究运用SELEX技术筛选获得的ssDNA核酸适配体H10和H13,具有高特异性和亲和性,可用于研发操作便捷、灵敏度高的哈维氏弧菌快速检测技术。     

核酸适配体氯化血红素比色法检测牛奶中的氯霉素

利用一种基于适配体氯化血红素(hemin)比色分析法快速检测牛奶中的氯霉素(chloramphenicol,CAP) 含量。设计两条DNA链,其中一条为CAP的核酸适配体,在其3′端修饰氯化血红素;另一条为CAP核酸适配体的互补链(cDNA),在其5′端修饰hemin。当体系中无CAP时,两条DNA单链杂交形成DNA双链,两个hemin单体形成二聚体使其过氧化物酶活性降低,以致不能催化过氧化氢氧化3,3′,5,5′-四甲基联苯胺(3,3′,5,5′-tetramethylbenzidine,TMB)产生蓝色产物。而体系中有CAP时,CAP与CAP适配体特异性结合,DNA双链解离,hemin单体的过氧化物酶活性恢复,并催化过氧化氢氧化TMB产生蓝色产物,加入反应终止液后,检测452 nm处的吸光度值。在优化条件下,该比色法检测CAP的线性范围为1～30 nmol·L-1,以3倍标准偏差计算其检出限为0.315 nmol·L-1。将该方法用于牛奶样品的检测,加标回收率为955%～1160%,表明该方法能够快速、准确和灵敏地检测牛奶中CAP含量。     

Magnetically assisted fluorescence ratiometric assays for adenosine deaminase using water-soluble conjugated polymers

A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deaminase assays with high sensitivity using water-soluble cationic conjugated polymers (CCPs). The assay contains three elements: a biotin-labeled aptamer of adenosine (biotin-aptamer), a signaling probe single-stranded DNA-tagged fluorescein at terminus (ssDNA-Fl) and a CCP. The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-Fl unhybridized, and the ssDNA-Fl is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field. In this case, after the addition of CCP to the magnetic beads solution, the fluorescence resonance energy transfer (FRET) from CCP to fluorescein is inefficient. Upon adding adenosine deaminase, the adenosine is converted into inosine, and the biotin-aptamer is hybridized with ssDNA-Fl to form doubled stranded DNA (biotin-dsDNA-Fl). The ssDNA-Fl is attached to the magnetic beads at the separation step, and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein. Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions. The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers. Supported by the “100 Talents” Program of the Chinese Academy of Sciences, and the National Natural Science Foundation of China (Grant No. 20574073)