炭疽芽孢杆菌芽孢适配子的结构与亲和性分析

为了对炭疽芽孢杆菌疫苗株A．16R芽孢的适配子进行亲和性分析,体外构建了78个碱基的随机DNA库,以炭疽芽孢杆菌疫苗株A．16R芽孢为靶标,用SELEX技术进行了15轮的筛,针室的适配子库进行克隆,测序,用Macaw2.05和DNAsisv2.5软件对每一适配子的保守序列和二级结构进行分析,并用生物素－链酶亲和素－辣根酶系统检测,根据OD值的高低判定亲和力的大小,对每一适配子进行亲和力测定。结果表明,适配子的亲和力高低不一,OD值最高为1．2,最低为0．25,二级结构分析结果显示,茎环和茎等二级结构可是适配子与芽孢结合的基础;其一级结构提示有AGGGG,CCCCG,GGCTT、ACACT等保守序,上述分类与OD值的高低分布基本吻合。     

寡核苷酸适配子在核医学中的应用

寡核苷酸适配子（aptamer，ataptable oligomer）是用指数富集配基的系统进化（SELEX）技术，通过体外筛选、扩增和富集获得能和小分子物质、多肽、蛋白质、细胞器、核酸及细胞和组织等靶物质高亲和、高特异结合的修饰寡核苷酸。适配子（aptamer）来源于拉丁语“aptus”一词，意为“使适合”。     

病理类型特异的卵巢癌组织适配子的鉴定

目的鉴定经组织切片消减SELEX筛选获得的4条候选适配子对不同类型卵巢癌组织的识别特性。方法公司合成、荧光素修饰候选适配子,利用免疫荧光实验对筛选获得的适配子候选序列进行鉴定。结果免疫荧光实验证明,适配子OC-10-19和OC-10-36能够特异识别卵巢浆液性乳头状囊腺癌,且OC-10-36还能够对早期的不典型增生进行识别,另2条适配子OC-10-10和OC-10-37仅识别个别的卵巢浆液性囊腺癌。结论成功筛选获得识别不同类型卵巢癌组织的特异适配子,为适配子用于不同病理类型标志物的鉴定、临床辅助诊断等提供了理论基础。     

炭疽芽孢杆菌防污染PCR-微孔板杂交-EIA检测技术的研究

目的 :建立防污染PCR_微孔板杂交_EIA检测技术 ,并用于炭疽芽孢杆菌芽孢的检测。方法 :根据炭疽芽孢杆菌毒力相关基因设计引物 ,筛选合适引物 ,建立用UDG酶防污染的PCR扩增_微孔板杂交与酶联显色检测炭疽芽孢杆菌的方法 ,并探讨试剂的室温稳定系统 ,将建立的方法用于模拟污染炭疽芽孢土壤样品的检测。结果 :根据炭疽芽孢杆菌荚膜和水肿因子基因设计的引物 ,可以同时检测两个毒力相关质粒的存在 ,用 0 .1U的UDG可以防止10 10 产物的污染 ,微孔板杂交_酶联显色检测比单纯PCR_电泳敏感 10倍。加入稳定剂的PCR_微孔板杂交_EIA体系可以耐受 7d的 37℃破坏试验。将该体系用于污染土壤的检测 ,可以检测出 0 .2 5g土壤中 10 3 个炭疽芽孢的存在。结论 :所研制的防污染PCR_微孔板杂交_EIA试剂克服了目前PCR试剂运输不便 ,产物污染所致假阳性和判定结果欠客观的缺点 ,为炭疽芽孢的快速检测提供了有力手段     

金纳米棒制备、修饰及与寡核苷酸适配子的耦联

目的金纳米棒的制备、修饰及与寡核苷酸适配子的耦联。方法利用种子调制生长法制备了金纳米棒,采用化学桥联方法对金纳米棒进行m-SH-PEG修饰及与靶向定位乳腺癌细胞的寡核苷酸适配子的耦联。结果成功制备了金纳米棒,以CTAB包裹的金纳米棒表面光滑、尺寸均一、分散性和稳定性良好;Zeta电位检测表明m-SH-PEG已成功修饰金纳米棒,琼脂糖电泳证实寡核苷酸适配子与金纳米棒成功耦联。结论成功地进行了金纳米棒的制备、修饰及与寡核苷酸适配子的耦联。     

噬菌体φ29末端酶大亚基gp16特异RNA适体的筛选

目的：筛选噬菌体φ29末端酶大亚基gpl6特异结合RNA适体。方法：采用SELEX技术，以微扎板为固定介质，在由25个碱基组成的随机区的RNA文库中进行筛选。结果与结论：经9轮筛选获得与gpl6特异结合的RNA适体，结合百分数从未经筛选的14.4％增加到第9轮的37．8％。测序结果大致分为5个家族。其二级结构多呈茎环或凸环结构。抑制性适体的筛选正在进行中。     

炭疽芽孢杆菌中和性抗体的研究进展

炭疽芽孢杆菌的中和性抗体在炭疽的被动免疫和治疗中有着重要的意义。抗保护性抗原（protective antigen,PA）抗体既能够抑制芽孢出芽又具有中和毒素的作用。抗致死因子（lethal factor,LE）/水肿因子（edema factor,EF）抗体能阻断致死毒素（lethal toxin,LT）/水肿毒素（edema toxin,ET）发挥活性,荚膜抗体能结合补体导致细菌繁殖体裂解。同时中和性抗体的水平也可以作为保护性免疫的评价标准。另一方面中和性单克隆抗体可以作为分析确定PA的中和性表位的有力工具。对中和性表位的分析既有利于探索炭疽毒素致病机制,也可以为现有炭疽疫苗的使用和发展表位疫苗等新型炭疽疫苗提供理论依据。该文综述了炭疽芽孢杆菌中和性抗体的研究进展。     

炭疽芽孢杆菌建立感染的过程及其机制

炭疽是一类烈性传染病,其病原体炭疽芽孢杆菌（简称炭疽菌）形成的芽孢是生物战剂和生物恐怖的原材料。免疫预防和特异性的治疗药物是应对这类生物威胁的重要手段。炭疽菌感染的机制研究,尤其是感染建立过程的研究能够为新型炭疽防治药物的研发提供新的思路。该文通过综述相关研究进展,介绍了炭疽菌的感染过程,描述了可能的感染机制,并结合炭疽防治药物的研究工作展开了讨论。     

炭疽芽孢杆菌A16R株无菌培养滤液的蛋白质组学分析

目的：初步分析由我国炭疽疫苗株A16R所生产的无菌培养滤液中的主要成分。方法：运用免疫蛋白质组学的手段,从炭疽A16R疫苗株无菌培养滤液的二维凝胶电泳中选择与保护性抗体结合和不与该血清结合但丰度高的蛋白质点,进行质谱鉴定和信号肽分析。结果：共选择73个点,从炭疽芽孢杆菌数据库中鉴定出66个。在66个点中,有43个点与保护性血清结合;23个为高丰度蛋白,不与该血清结合。在与保护性血清结合的43个点中,13个点为不同大小的PA分子,其中相对分子质量83×10^3和63×10^3占主导,相对分子质量较小的片段仅4个。其余成分包括炭疽表面蛋白EA1、Sap、胶原黏附蛋白、伴侣分子DnaK等。结论：我们制备的炭疽无菌培养滤液中含有PA成分,PA是炭疽疫苗的重要保护性抗原。但PA以外的其他成分在抗炭疽免疫中起何种作用,是否参与诱发机体免疫应答有待进一步研究。     

炭疽芽孢杆菌S-层蛋白功能研究进展

炭疽芽孢杆菌是人畜共患病炭疽的病原菌。目前,对炭疽杆菌2个毒力大质粒（编码主要毒力基因的pXO1与编码合成荚膜基因的pXO2）的研究比较深入;炭疽杆菌细胞壁与荚膜间还存在一种蛋白性质的类晶体（paracrystalline）结构：S-层（surface-layer,表层）结构。炭疽杆菌的S-层蛋白主要为表面排列蛋白（surface array pro-tein,Sap）和胞外抗原1（extracellular antigen 1,EA1）,此外,在炭疽杆菌中还存在其他一些与S-层相关的蛋白,了解这些蛋白的相互作用及免疫机制对深入认识炭疽杆菌的致病机制具有重要意义。本文将就近年来关于炭疽杆菌S-层蛋白成分、与细胞壁的连接、S-层基因的调控、致病性及其与宿主免疫机制的关系等方面的研究进展做一简要综述。     

Comparison of noninvasive sampling sites for early detection of Bacillus anthracis spores from rhesus monkeys after aerosol exposure

Bacillus anthracis, a spore-forming bacterium, is the etiologic agent of anthrax. B. anthracis spores can be aerosolized, are relatively easy to produce, and are capable of producing high mortality when inhaled. The prompt use of postexposure antibiotics combined with vaccination greatly increases the survival rate. Rapid detection of exposure is critical to effective case management. Using common collection swabs, culture medium, and culturing equipment, we compared six different noninvasive sampling sites to determine which might best be used to rapidly detect the presence of B. anthracis spores on rhesus monkeys after aerosolization. The results indicate that the greatest number of spores were deposited in the nares, on the face, and on the haired portions of the head, suggesting that these locations are the most effective sampling sites when attempting to detect B. anthracis aerosol exposure.     

基于毛细管电泳的细胞SELEX方法的初步建立

目的：初步建立一种基于毛细管电泳技术的细胞指数式富集的配基系统进化（SELEX）技术,实现快速、高效和微量筛选。方法：以表达有绿色荧光蛋白的HepG2细胞作为靶细胞,FITC标记的DNA文库作为初始文库,采用带有激光诱导荧光检测器的毛细管电泳（CE）进行检测分离,收集制备次级文库。同时采用常规方法进行了一轮细胞筛选,以进行比较。结果及结论：流式分析结果显示与常规方法相比,引入毛细管电泳分离技术后经过一轮筛选,特异配基即获得了较好的富集。这表明基于毛细管电泳的细胞SELEX方法获得了初步建立,为实现肿瘤细胞的快速微量筛选奠定了基础。     

Comparison of real-time polymerase chain reaction and conventional polymerase chain reaction methods for the rapid identification of Bacillus anthracis

Bacillus anthracis spores have been shown to be one of the most effective biological weapons. For the rapid detection of B. anthracis spores, several genetic markers, including chromosomal and plasmid-based sequences, were studied with polymerase chain reaction (PCR) methods. In the present study, a method using a primer/probe set based on the pXO1-encoded pag gene for the detection of B. anthracis was tested in addition to culture. Eight pathological samples (four blood-immersed cotton specimens, two spleen tissue specimens, and two blood smears) with confirmed positive results for anthrax were used. All samples were suspended in saline solution and fixed with Gram and Giemsa stains for examination of colony and capsule formation. Amplicons were analyzed on 2% agarose gels with the classic PCR method. For real-time PCR, a fluorescently labeled TaqMan probe was used with a Smartcycler. Positive smear and cotton samples were confirmed with the standard culture and real-time PCR methods, but the same samples were found to be negative with the classic PCR method. A spleen sample known to be positive for B. anthracis was found to be negative with the culture method because of possible contamination with Proteus-type bacteria.     

定量PCR快速检测炭疽芽孢杆菌的实验研究

目的建立一种简便、快速、定量检测炭疽芽孢杆菌的PCR方法。方法采用TaqMan荧光探针技术,针对炭疽芽孢杆菌质粒pXO1、pXO2上的基因设计引物;用炭疽芽孢杆菌（Sterne株）污染环境土壤来模拟实际样本,比较了从土壤中提取DNA的不同方法对检测效果的影响,并与同类进口试剂进行了平行比对。结果以含有检测目的片段的线性质粒为模板,反应体系的灵敏度可达到101拷贝/μl;用Sterne株芽孢悬液污染土壤,检测灵敏度可达2.5×103cfu/g土壤。我们研制的试剂与进口试剂在敏感性和准确性方面一致,且操作简便。结论本研究建立的炭疽芽孢杆菌实时定量PCR检测方法可特异、灵敏地检测土壤标本中可疑目标菌。     

炭疽恐怖袭击直接经济损失评估方法

以炭疽杆菌芽孢气溶胶恐怖袭击为例,对城市地区遭受非传染性生物剂攻击的直接经济损失评估方法进行研究.将经济损失分为死亡损失、医疗损失、环境洗消、经济活动停顿4个方面构建评估模型,最后以北京某商业区为例,对遭受炭疽杆菌芽孢气溶胶恐怖袭击的经济损失进行计算和分析.相关评估方法可以对生物恐怖袭击后政府资源调配、经济援助等决策提供支持和依据.     

Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents

Two DNA aptamers directed against two separate exosites on human alpha-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.     

Extending the lifetime of anticoagulant oligodeoxynucleotide aptamers in blood

We have investigated (123)I and (125)I DNA aptamer analogs of anticoagulant DNA aptamers to thrombin exosite 1 and exosite 2 for thrombus imaging potential. Two severe problems are rapid clearance from circulating blood and blood nuclease. With aptamers (unlike antisense) the nucleotide analogs used in polymerase chain reaction-selection cycles also must be used in the radiotracer. We investigated 3'-biotin-streptavidin (SA) bioconjugates of the aptamers to alleviate these problems. Blood nuclease assays and biodistribution analysis were used in the mouse and rabbit. We found that 3'-biotin protected the aptamers significantly from blood nuclease in vitro, but it did not slow in vivo clearance. In contrast, the 3'-biotin-SA bioconjugates were resistant to blood nuclease in vitro and were also longer-lived (10-20 times) in vivo. Bioconjugate aptamers retained affinity for thrombin. Two solutions emerge: 1) In noncirculating blood (within a thrombus) 3'-biotin extends aptamer lifetime, whereas 2) in circulating blood (the transport medium), where more aggressive clearance is encountered, 3'-SA extends aptamer lifetime.     

靶标诱导变构解离SELEX技术筛选皮质醇特异核酸适体

目的通过指数富集配体系统进化（SELEX）技术获得皮质醇特异性核酸适体,建立高品质、超敏、易用和稳定的皮质醇检测试剂盒。方法人工合成两端固定序列,中间为35 bp随机序列,生成80 bp长度ssDNA文库,捕获序列固定在磁珠上,文库与其退火组装,皮质醇的特异核酸适体通过我们建立的靶标诱导变构解离SELEX（TISSD-SELEX）技术获得。最后一轮的PCR产物经克隆测序,MegAlign和RNAstructure软件分析其一级和二级结构。实时（real-time）apta-PCR方法分析G12的亲和活性。结果经过12轮筛选后,获得10个皮质醇的核酸适体。序列分析显示,特异核酸适体以高G含量核酸序列为主,软件预测其中8个序列主要以G四联体二级结构为主,候选核酸适体G12显示了可以作为生物探针用于皮质醇检测。结论成功建立TISSD-SELEX技术,获得小分子靶标皮质醇核酸适体。小分子靶标无需将其偶联在固相介质或大分子上,对靶分子结构、性质无特殊要求,因而有望成为小分子靶标核酸适体筛选的通用技术方法。     

特异识别已分化PC12细胞的ssDNA适体的结构和功能研究

目的：研究特异识别已分化PC12细胞的ssDNA适体的二级结构及截短前后的适体与已分化PC12细胞的结合能力。方法：利用RNA structure3．5软件对截短前后的适体的空间结构进行模建，体外合成这些截短适体，利用流式细胞术和荧光显微镜研究适体与已分化PC12细胞的结合能力。结果：单茎环结构是适体与已分化PC12细胞结合的二级结构；截短后的适体与已分化PC12细胞的结合能力明显大于与未分化PC12细胞结合能力；AP17-38与已分化PC12细胞结合的荧光强度明显大于与未分化PC12细胞结合的荧光强度；截短前后的适体均能够特异识别混合物中已分化PC12细胞。结论：截短后的适体能够保留适体的空间结构，而且与已分化PC12细胞的亲合力不会降低。