羧酸化环糊精复合的银纳米簇的合成及其抗菌能力测试

本研究以羧甲基-β-环糊精（CM-β-CD）为模板合成 AgNCs,探讨了 AgNCs 的合成条件,对其进行了表征,并对其抗菌能力进行了研究。结果显示,当溶液的 pH 值为5．98,CM-β-CD 和 AgNO3的比例为1∶1时,合成的银纳米簇荧光强度达到最大。以大肠杆菌为研究对象,对环糊精合成的银纳米簇进行抗菌实验测试,发现由于银纳米簇比表面较大,表面活化能高使其比银离子和银溶胶具有更好的抑菌能力。     

前体物对石蒜悬浮细胞生长和生物碱积累的影响

为探究苯丙氨酸、酪氨酸和酪胺3种前体物对石蒜悬浮细胞系生长和生物碱积累的影响。通过向培养基添加不同浓度的3种前体物,以及同时添加苯丙氨酸和酪氨酸,考察其对细胞生长量及细胞中生物碱累积的影响。结果表明:苯丙氨酸对细胞的生长和生物碱的积累影响不明显;酪氨酸和酪胺作用显著:添加200μmol/L酪氨酸,细胞中生物碱的含量是对照组的2.56倍,其中力可拉敏和加兰他敏含量为3.77 mg/g和4.46 mg/g,分别是对照组的6.61倍和6.97倍;添加200μmol/L酪胺,细胞中生物碱含量是对照组的2.63倍,力可拉敏和加兰他敏含量为4.45 mg/g和5.14 mg/g分别是对照组的9.08倍和9.18倍;在200μmol/L酪氨酸的基础上添加苯丙氨酸没有明显的增效作用。表明添加酪氨酸和酪胺对细胞生长及生物碱生物合成具有显著的促进作用     

磷酸酪氨酸蛋白专一抗体的制备和纯化

以偶联磷酸化酪氨酸的牛血清白蛋白（ＢＳＡ）作为免疫原免疫兔获得抗血清．自抗血清中分离获得抗体．自酪胺合成磷酸酪胺,并偶联到溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ上．抗体经磷酸酪胺－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化,所得抗体专一性强,Ｄｏｔｂｌｏｔ显示：抗体仅对酪氨酸磷酸化的蛋白质包括酪氨酸磷酸化的血清白蛋白,溶菌酶,卵清蛋白起抗原抗体反应,而不识别非酪氨酸磷酸化的溶菌酶,卵清蛋白,也不识别作为免疫原的骨架成分ＢＳＡ,也不识别丝氨酸磷酸化的卵清蛋白和苏氨酸磷酸化的卵清蛋白．     

抗HBsAg-碱性磷酸酶双功能抗体分子的构建

为构建带有碱性磷酸酶活性的双功能基因工程抗体, 用PCR方法克隆大肠杆菌碱性磷酸酶基因, 通过酶切分析和DNA序列测定核实后,将其重组到抗乙肝表面抗原(HBsAg) Fab段的Fd羧基端,构建重组融合蛋白表达载体pHBFAP, 转化大肠杆菌XL1-Blue, 经异丙基硫代-β-D-半乳糖苷诱导表达后, 采用ELISA法检测到培养上清中存在与HBsAg的结合活性和碱性磷酸酶的催化活性, 显示抗HBsAg-碱性磷酸酶双功能抗体分子在大肠杆菌中获得了表达.     

氨基葡萄糖对酪氨酸酶的抑制作用

通过对酪氨酸酶催化底物L-DOPA反应速率的观察测定,研究了氨基葡萄糖（G-NH2）对酪氨酸酶的抑制作用。在反应液中加入50μL浓度为2.2 mg/mL G-NH2时（体系中G-NH2终浓度为36μg/mL）,酶抑制率为50%。GNH2对酪氨酸酶的抑制作用是个复杂的过程,酶反应呈先促进后抑制。分析酶抑制曲线Lineweaver-Burk双倒数图,得出G-NH2为混合抑制剂,进一步研究发现多巴醌生产量会减少,抑制类型是不可逆抑制。     

酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法的建立

目的:建立一种酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法,提高基因芯片检测的灵敏度。方法:待测靶基因与固定在玻片上的探针杂交,依次加入链霉亲和素标记的辣根过氧化物酶、生物素标记的酪胺及链霉亲和素标记的量子点,37℃孵育,然后加入银增强试剂显色,最后用可视化生物芯片扫描仪扫描并记录结果;以牛布鲁菌210105株为检测对象,以酪胺信号放大-荧光素Cy3(TSA-Cy3)检测法为对照方法,测定酪胺信号放大-量子点标记银染增强(TSA-QDS)检测法的灵敏度。结果:确定了基因芯片量子点标记银染增强可视化检测方法的检测流程,优化了检测条件,并考察了检测灵敏度。优化的检测条件为:酪胺-生物素稀释比例为1∶4000,链酶亲和素标记的量子点稀释比例为1∶50,37℃孵育时间为25~30 min,银染增强时间为6~7 min。检测牛布鲁菌的灵敏度为103CFU/mL。结论:该方法实现了基因芯片高灵敏度可视化检测,其灵敏度与荧光法相当,并且有可视化的优势。     

长角血蜱唾液腺中腺苷三磷酸双磷酸酶的纯化及其酶切机制

利用染料亲和层析(Cibacorn Blue柱)和离子交换层析(Macrosphere WCX柱)对长角血蜱Haemaphysalis longicornis唾液腺的腺苷三磷酸双磷酸酶进行纯化,经SDS-PAGE证实其分子量为66 kD。腺苷三磷酸双磷酸酶可以水解ATP和ADP,但对AMP无水解作用,水解ATP和ADP的K_m值均为0.2 μmol/L,V_max值分别为12.5和15.6 μmol/(min·mg)。腺苷三磷酸双磷酸酶水解ATP的中间产物是ADP,最终产物是AMP和正磷酸。表明腺苷三磷酸双磷酸酶水解ATP的位点是5'-核苷酸的γ-磷酸键,水解ADP的位点是5'-核苷酸的β-磷酸键。     

MC 3T3-E1细胞在纳米羟基磷灰石/壳聚糖复合支架上的增殖和分化

用原位合成纳米羟基磷灰石的方法制备多孔纳米羟基磷灰石/壳聚糖复合支架;在支架上接种MC3T3-E1细胞,瑞氏染色检测细胞形态,MTT法检测其增殖情况;在诱导培养基中培养30d后,碱性磷酸酶染色比较其分化水平;定量检测细胞的碱性磷酸酶活性;RT-PCR检测成骨相关基因的表达情况。实验结果表明:MC3T3-E1细胞在纳米级羟基磷灰石/壳聚糖复合支架上粘附铺展良好,其增殖率显著高于培养于纯壳聚糖支架上的细胞。碱性磷酸酶染色表明复合支架上的细胞有较高水平的碱性磷酸酶表达。进一步定量检测细胞的碱性磷酸酶活性,结果说明在复合支架上细胞比纯壳聚糖支架上培养的细胞碱性磷酸酶活性提高了约8倍。此外,骨分化相关特征基因骨桥蛋白OPN在复合支架上培养的细胞中的表达水平也明显高于纯壳聚糖上培养的细胞。分化成熟标志基因骨钙素OC在复合支架上培养的细胞中有表达,但是纯壳聚糖支架上培养的细胞中却未检测到。支架中纳米羟基磷灰石的加入不仅提高了前成骨细胞在复合支架上的增殖,而且还促进了它的分化。纳米羟基磷灰石/壳聚糖复合支架表现出良好的生物相容性和生物活性,是极具前景的骨组织工程支架材料。     

白花银背藤中的生物碱及其细胞毒活性研究

为研究白花银背藤(Argyreia seguinii)的生物碱类成分及其细胞毒活性，该研究采用硅胶、ODS、Sephadex LH-20及半制备HPLC等柱色谱法对白花银背藤的75%乙醇提取物进行分离纯化，根据理化性质及波谱数据鉴定化合物的结构，并通过MTS法检测化合物对5种肿瘤细胞的增殖抑制活性。结果表明：从白花银背藤中分离得到9个生物碱类化合物，分别鉴定为N-反式桂皮酰对羟基苯乙胺(1)、N-反式对香豆酰基酪胺(2)、N-反式阿魏酰酪胺(3)、N-顺式阿魏酰酪胺(4)、1H-吲哚-3-甲醇(5)、金色酰胺醇酯(6)、8-氧-四氢巴马亭(7)、8-oxypalmatine(8)和12,13-dihydro-8-oxoberberine(9)。其中，化合物1、4-9均为首次从该植物中分离得到，化合物7和化合物8对肝癌SMMC-7721细胞显示细胞毒活性，半抑制浓度(IC₅₀)值分别为(15.50±0.76)、(14.24±0.72)μmol·L^-1。该研究结果为进一步探讨白花银背藤的化学成分和药理活性奠定了基础，为厘清壮药“一匹绸”的药材基原...     

DNA甲基化作用对HL-60细胞中蛋白质-核酸相互作用的影响

以S-腺苷酰-L-甲硫氨酸(SAM)为诱导物,在10μmol/L的最佳浓度下,可诱导16%的HL-60细胞分化.HPLC法检测碱基含量,发现在细胞分化过程中伴有基因组DNA甲基化水平升高.选择对5-甲基胞嘧啶敏感的限制性核酸内切酶切割DNA,证实基因组DNA对HaeⅢ,SmaⅠ,SalⅠ,XhoⅠ和HindⅢ的切割产生阻抗作用.以凝胶滞留法检测DNA与核蛋白的结合状况,表明DNA与胞内DNA结合蛋白的结合能力发生改变.     

一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

[背景]碱性磷酸酶作为工具酶被广泛应用于各个领域,在免疫学检测方面应用较多的是PhoA家族的碱性磷酸酶,尚无关于PhoD家族的碱性磷酸酶在免疫学检测方面的研究。[目的]筛选出一株产高酶活性PhoD家族碱性磷酸酶的细菌,并将其phoD基因进行克隆表达,研究PhoD的酶学性质,为PhoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。[方法]采取有机质丰富的土样在有机磷平板中进行细菌分离,以4-硝基苯磷酸二钠盐（4-nitrophenyl phosphate disodium salt hexahydrate,p-NPP）为底物测定有机磷平板中单菌落的酶活性,选取酶活性高的菌株作为目的菌株,克隆其phoD基因。[结果]筛选到一株产碱性磷酸酶酶活性高的菌株S2-4,通过16S rRNA基因序列同源性比较分析,鉴定该菌株为解淀粉芽孢杆菌,克隆了其phoD基因并进行诱导表达。研究了纯化后PhoD的酶学性质,PhoD的最适反应温度为70℃;最适反应pH为9.8;PhoD最适Ca²⁺浓度为3 mmol/L,Mg²⁺对PhoD的酶活性有抑制作用,K     

A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters

A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer‐templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA–AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as‐prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA–AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Inhibition of DNA replication initiation by silver nanoclusters

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.     

Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.     

Toward site-specific, homogeneous and highly stable fluorescent silver nanoclusters fabrication on triplex DNA scaffolds

A new strategy to create site-specific, homogeneous, and bright silver nanoclusters (AgNCs) with high-stability was demonstrated by triplex DNA as template. By reasonable design of DNA sequence, homogeneous Ag(2) cluster was obtained in the predefined position of CG.C(+) site of triplex DNA. This strategy was also explored for controlled alignment of AgNCs on the DNA nanoscaffold. To the best of our knowledge, this was the first example to simultaneously answer the challenges of excellent site-specific nucleation and growth, homogeneity and stability against salt of DNA-templated AgNCs.     

Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.     

高盐沉淀CTAB法提取温室菊花基因组DNA

根据温室菊花植物组织富含多酚、多糖的具体特性,对CTAB法加以改进:在待沉淀液中加入1/2体积5 mol·L～NaCI.改进后的方法获得的DNA质量良好,电泳条带清晰,提取过程无明显的DNA降解,基本上排除了多酚物质的干扰.以提取的DNA为模板,用一对引物扩增菊花中18S基因,得到条带单一,大小与已知一致,说明获得的DNA可以进行PCR扩增,EcoR I 酶切基因组DNA图谱表明,提取的DNA能被限制性内切酶完全酶切,可以满足相关的分子生物学研究.     

美国黑核桃SSR反应体系优化

优化SSR-PCR反应体系是黑核桃（Juglans nigra L.）SSR基因鉴定和群体遗传等研究的基础。本研究通过对PCR反应中Mg²⁺浓度、牛血清白蛋白（Bovine Serum Albumin,BSA）浓度、Taq聚合酶用量、dNTPs浓度、引物浓度和模板DNA量的组合以及PCR程序组合试验,确定了黑核桃SSR的最佳反应体系,即在10 μL的PCR反应体系中,含10 ng模板DNA,0.1 mg·mL^-1牛血清蛋白（BSA）,0.25 mmol·L^-1 dNTPs,1.5 mmol·L^-1 Mg²⁺ 1 μL 10X Taq DNA聚合酶反应缓冲液,0.5 U Taq聚合酶,1.0 mmol·L^-1单对引物（0.5 mmol·L^-13对引物）。SSR-PCR反应扩增程序为：94℃变性3 min;93℃变性15 s,50℃或者53.5℃退火1 min,72℃延伸30 s,32个循环;72℃后延伸10 min,置4℃保存。利用此反应体系对黑核桃进行PCR扩增并电泳检测,其结果清晰、稳定、可靠,适合进一步对黑核桃群体遗传、基因型鉴定和分子生态研究。     

光萼荷属植物SSR反应体系确立与指纹图谱构建

利用正交试验设计优化并确立光萼荷属（Aechmea）植物SSR反应体系,构建部分光萼荷属植物的SSR分子指纹图谱。结果表明：光萼荷属植物的最佳SSR反应体系为10 μL总体积包括1×PCR buffer、Mg²⁺ 2.0 mmol·L^-1、dNTPs 200 μmol·L^-1、引物2.5 μmol·L^-1、模板DNA 90 ng和Taq DNA聚合酶1.5 U。利用该体系和6对SSR引物对15份光萼荷属植物进行扩增反应和电泳检测,其中M1、M3、M4等3对SSR引物扩增图谱清晰、多态性较高,均能将该15份光萼荷属植物鉴别出来,初步建立了15份光萼荷属植物的SSR分子指纹图谱,进一步证明该SSR反应体系稳定可靠,可以有效用于光萼荷属植物种质资源鉴定。     

A reversible DNA-silver nanoclusters-based molecular fluorescence switch and its use for logic gate operation

Molecule-like silver nanoclusters (AgNCs) with few to tens of atoms are highly sensitive to the sequence and structure of DNA stabilizers. In this paper, a novel pH-triggered reversible molecular fluorescence switch is developed by taking advantage of the DNA-dependent fluorescence pH response of AgNCs. The DNA-AgNCs fluorescence switch simultaneously addresses concerns of simple construction strategy, efficient design and organic-solvent-free operation. Moreover, the excellent photostability and biocompatibility of AgNCs provide great potential for application of the DNA-AgNCs fluorescence switch in the development of functional molecular devices. Specifically, we apply the DNA-AgNCs fluorescence switch combined with the DNA sequence-dependent pH response pattern of AgNCs for construction of molecular logic gates.