工业大麻种子萌发及渗透胁迫下实时荧光定量PCR内参基因的筛选

为了筛选适用于工业大麻种子正常萌发与渗透胁迫下萌发的实时荧光定量PCR(qRT-PCR)内参基因,研究以‘云麻7号’种子为试验材料,设置正常萌发和20%PEG渗透胁迫下萌发处理.利用qRT-PCR技术对10个候选参考基因（CDPK,e IF4E,TIP41,UBQ,EF1α,PP2A,Actin,Clathrin,TUB,DHS2）在3个萌发时间点（萌发3、5、7 d）幼苗中的表达量进行检测.通过GeNorm、Normfinder和BestKeeper三种软件包对内参基因表达稳定性进行评价,再利用RefFinder在线软件进行综合分析,从而获得稳定、适宜的内参基因.结果表明,在工业大麻种子正常萌发过程中,PP2A基因的稳定性最好.在渗透胁迫下的种子萌发中,UBQ基因的稳定性最好.综合两组结果得出,eIF4E内参基因的表达最稳定.研究结果确定了工业大麻种子萌发及渗透胁迫下进行qRT-PCR分析的最适内参基因,为今后研究工业大麻种子萌发响应渗透胁迫相关基因的表达提供前期基础.     

中华鳖实时定量PCR内参基因的筛选与评价

选择合适的内参基因是采用q-PCR方法研究基因表达的前提.选取中华鳖GAPDH、EF1α、18SrRNA、Tubulin和β-actin等5个候选内参基因,通过q-PCR方法得到各基因在不同组织中的Ct值,利用4种内参筛选软件和综合评定法进行评价.geNorm和NormFinder软件分析均显示18SrRNAEF1αβ-actinGAPDHTubulin(稳定性由高到低).BestKeeper软件分析显示18SrRNAGAPDHEF1αβ-actinTubulin.RefFinder软件分析和综合评价法显示18SrRNA最佳.推荐优先使用18SrRNA或EF1α,不建议用β-actin和Tubulin.上述结果可为中华鳖和其它爬行动物的基因表达研究提供参考依据.     

A/PR8/34(H1N1)NA基因包装信号对H5N1流感疫苗株NIBRG-14产量的影响

NIBRG-14是采用"6+2"策略制备的一株H5N1灭活疫苗株,其表面抗原HA和NA基因来自于A/Vietnam/1194/2004(H5N1,VN1194),内部基因来自于A/Puerto Rico/8/34(H1N1,PR8),已有研究表明该疫苗株在鸡胚中的产量不佳.本研究发现,在PR8背景下,VN1194NA基因被包装入重组病毒中的效率仅为正常包装量的38%～68%,因此有一部分重组病毒为不含有NAvRNA的缺陷型病毒粒子.本研究通过在VN1194NA基因完整编码区(CDS)的5′和3′两端嵌合PR8NA基因包装信号序列(vRNA3′末端41bp,5′末端67bp)的方法,使重组病毒中NAvRNA的包装效率得到完全恢复,并且病毒在鸡胚的生长滴度提高了10倍,血凝素HA含量提高了约2·7倍,从而为H5N1流感疫苗株的研制提供了新的思考方向.     

授粉后红球姜雌性生殖器官qRT-PCR的内参基因筛选

在研究分析红球姜（Zingiber zerumet (L.) Smith）败育关键调控基因的表达中，筛选授粉后雌性生殖器官发育过程中的内参基因至关重要。本研究根据授粉后不同时间点的红球姜转录组数据库以及相关文献报道的传统内参基因，筛选出10个表达相对稳定的基因Actin-2（ACT2）、Actin-7（ACT7）、Beta tubulin-1（TUB1）、Beta tubulin-5（TUB5）、Alpha tubulin-3（TUA3）、Ubiquitin（UBQ）、Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）、Elongation factor 1-alpha（EF-1）、Cyclophilin（CYP）、Histone（H2A）作为候选内参基因，采用qRT-PCR技术，结合GeNorm、NormFinder和BestKeeper软件对候选内参基因的表达稳定性进行分析。结果表明，在红球姜雌性生殖器官授粉后的发育过程中，GAPDH和UBQ的表达稳定性最好，均适合作为内参基因，且同时使用两种作为内参基因能使实时荧光定量PCR标准化分析结果更精确。因此，最终选择GAPDH和UBQ作为实时荧光定量PCR标准化分析红球姜雌性生殖器官相关基因表达的内参基因。本研究将为探究红球姜败育的分子机理奠定基础，也为近源姜属植物内参基因的筛选提供线索。     

无机-有机杂化钼磷、钒钼磷多酸化合物的合成与结构

通过对V2 O5 ,Na2 MoO4·2H2 O ,HF ,Ni(NO3) 2 ·6H2 O ,H3PO4和吡啶体系水热反应的研究 ,合成并表征了无机 -有机杂多酸化合物 [C5 H6 N] 3[C5 H5 N][PMo12 O40 ] ( 1 ) ,[C5 H6 N] 4[PVMo11O40 ] ( 2 )和 [C5 H6 N] 4[HPV2 Mo10 O40 ] ( 3 ) .化合物( 1 )属单斜晶系 ,C/ 2c空间群 ,a =2 .1 867( 6)nm ,b =1 .3 978( 4)nm ,c =1 .663 3( 3 )nm ,β =1 0 5.73 ( 1 0 )°,V =4 .893nm3,R =0 .0 60 4 ,Z =4 ;化合物 ( 2 )属单斜晶系 ,C/ 2c空间群 ,a =2 .1 858( 4)nm ,b =1 .3 971 ( 3 )nm ,c =1 .663 5( 3 )nm ,β =1 0 5.83 ( 3 )°,V =4 .8873 ( 1 7)nm3,Z =4 ,R =0 .0 4 63 ;化合物 ( 3 )属单斜晶系 ,P2 1n空间群 ,a =1 .1 1 2 2 ( 2 )nm ,b =1 .94 1 2 ( 3 )nm ,c=1 .2 4 84 ( 3 )nm ,β =99.89( 3 )°,V =2 .6565( 9)nm3,Z =2 ,R =0 .0 690 .     

锌三元配合物的合成及其抑菌活性研究

以反丁烯二酸及3,5-二(3-吡啶基)-1,2,4-三氮唑为原料,用水热法合成配合物[Zn2(C12H8N5)2(C4H2O4)(H2O)2]n(C12H8N5=3,5-二(3-吡啶基)-1,2,4-三氮唑,C4H2O4=反丁烯二酸)。X-射线单晶结构分析表明,该配合物属单斜晶系,空间群为P21/n,晶胞参数为a=1.259 1(3)nm,b=0.776 10(16)nm,c=1.450 1(3)nm,β=94.00(3)°,Z=4。在配合物中,配体3,5-二(3-吡啶基)-1,2,4-三氮唑以双齿配位的形式参与配位,其通过吡啶环上的N原子以及三氮唑环上的氮原子桥连2个锌离子;反丁烯二酸2个羧基氧原子分别与2个双核锌单元中的2个锌离子桥连;中心金属离子Zn(Ⅱ)与来自反丁烯二酸羧基的1个氧原子、2个3,5-二(3-吡啶基)-1,2,4-三氮唑的2个氮原子以及1个水分子的氧原子配位,配位数为4;中心金属离子Zn(Ⅱ)与4个配位原子形成一个畸变的四面体构型。体外抗菌试验结果表明,配体和配合物对金黄色葡萄球菌、大肠杆菌、枯草杆菌均有中等抑菌效果。     

烷基氮氧自由基推拉效应的ab initio研究--I.烷基氮氧自由基的ESR波谱

研究了烷基氮氧自由基的 ESR波谱。结果表明 :室温条件下 H2 NO· ,CH3NHO· ,(CH3) 2 NO·和 (C2 H5) 2 NO·自由基均能产生 5条、7条、2 1条和 1 3条偶合分裂峰 ,相应的 H原子和 N原子与自由基单电子之间的偶合分裂常数分别为 :αH=1 1 .9,αN=1 1 .9(CH3OH中 ) ;αH=1 3.8,αN=1 3.8(CH3OH中 ) ;αH=1 2 .3,αN=1 5.2 (CCl4 中 ) ;αH=1 0 .3,αN=1 5.2 (CCl4 中 ) ;同时 ,讨论了 (C2 H5) 2 NO·在不同有机溶剂中的偶合分裂常数及可能的衰变机理     

一个基于5-(2-吡啶基)-3,3′-联(1H-1,2,4-三氮唑)和均四苯甲酸的锌(II)超分子配合物的合成、晶体结构与荧光性质(英文)

在单核化合物[Zn(C9H7N7)(C10H4O8)(H2O)3]·3H2O中,Zn(II)金属中心离子分别和4个氧和2个氮原子配位,其中4个氧原子来自于一个去质子的均四苯甲酸(H2BTCA)和3个配位水分子,氮原子来自5-(2-吡啶基)-3,3'-联(1H-1,2,4-三氮唑)(H2pbt)配体.在这个结构中,相邻的单核单元之间通过分子内和分子间的O-H···O和NH···O氢键作用连接成二维超分子结构,且三唑环与苯环之间以及相邻吡啶环之间存在芳香堆积作用.此外,还研究了标题配合物的固态荧光性质.     

三株H1N1亚型猪流感病毒M基因序列测定与进化分析

正猪流感,是集约化养猪场普遍存在且难以根除的猪呼吸道疾病之一,也是世界上最常见的猪传染病之一,目前有10种血清亚型。H1N1亚型SIV在中国日趋复杂和多变,与其他亚型流感病毒密切相关,并在其他谱系之间发生多重重组,其中基质蛋白位于流感病毒第7段,含有1 027个核苷酸,具有M1和M2两个相互重叠的编码区。本试验通过对三株H1N1亚型SIV的M基因进行鉴定分析,为进一步防控猪流感病     

稀土配合物Nd(C5H8NO3)2(C3H5N2)2Cl3·4H2O的合成、表征及性能研究

合成了稀土氯化钕与N-乙酰-DL-丙氨酸(C5H9NO3,Ac-Ala)及咪唑(C3H4N2,Im)的三元配合物,通过元素分析和化学分析确定其化学组成为Nd(C5H8NO3)2(C3H5N2)2Cl3*4H2O,并通过FTIR、UV、1H NMR、13C NMR和X-射线粉末衍射的等手段对目标配合物的结构进行了表征.根据DSC和TG-DTG曲线研究了配合物的热分解过程.配合物水溶液的荧光性质测试表示,形成配合物后,配合物水溶液发生了荧光淬灭现象,在10-2～10-4 mol/L浓度范围内荧光强度与溶液浓度呈正相关.     

H5N1亚型禽流感病毒血凝素基因的克隆与表达

 根据已知H5N1亚型禽流感病毒血凝素(HA)基因序列设计、合成克隆引物.自灭活的云南地方H5N1亚型病毒阳性临床组织样品中提取总RNA,反转录后采用高可信度DNA聚合酶(Pyobest^TMDNA Polymerase)扩增HA基因,采用Invitrogen定向表达系统(Champion^TMpET directional TOPO expression system)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组HA,分子质量约78ku.采用阳性血清经免疫印迹及ELISA分析重组HA的免疫反应性,结果表明重组HA能与H5N1亚型病毒抗血清发生特异性结合,具有良好的免疫反应性.     

禽流感病毒H5N1亚型RNA聚合酶蛋白表达纯化及晶体生长

通过使用原核表达载体大量表达H5N1病毒RNA聚合酶亚基PA-C257,PB1_N25,再经过GST亲和层析和Sephadex G-200层析柱纯化,获得了高纯度的蛋白复合体.采用悬滴气相扩散法筛选蛋白晶体,在1～1.5mol/L乙酸钠和pH7.9条件下获得了理想的晶体,为解析禽流感病毒RNA聚合酶三维结构并进一步认识其生物功能奠定了基础.     

Genome evolution of novel influenza A （H1N1 ） viruses in humans

The epidemic situation of A H1N1 flu arose in North America in April 2009, which rapidly expanded to three continents of Europe, Asia and Africa, with the risk ranking up to 5. Until May 13th, the flu virus of A H1N1 had spread into 33 countries and regions, with a laboratory confirmed case number of 5728, including 61 deaths. Based on IRV and EpiFluDB database, 425 parts of A H1N1 flu virus sequence were achieved, followed by sequenced comparison and evolution analysis. The results showed that the current predominant A H1N1 flu virus was a kind of triple reassortment A flu virus： （i） HA, NA, MP, NP and NS originated from swine influenza virus; PB2 and PA originated from bird influenza virus; PB1 originated from human influenza virus. （ii） The origin of swine influenza virus could be subdivided as follows： HA, NP and NS originated from classic swine influenza virus of H1N1 subtype; NA and MP originated from bird origin swine influenza virus of H1N1 subtype. （iii） A H1N1 flu virus experienced no significant mutation during the epidemic spread, accompanied with no reassortment of the virus genome. In the paper, the region of the representative strains for sequence analysis （A/California/04/2009 （H1N1） and A/Mexico/4486/2009 （H1N1）） included USA and Mexico and was relatively wide, which suggested that the analysis results were convincing.     

H1N1 流感病毒感染小鼠在免疫学研究中的初步应用

摘要: 目的研究H1N1 流感病毒感染能力及机体免疫应答反应。方法利用0. 1LD50剂量A/PＲ/8 /34 ( H1N1) 病毒感染C57BL/6 小鼠,利用ELISA 和流式细胞术等方法检测IL-5、IL-6 水平和CD4 + 效应性T 淋巴细胞比例。结果3dpi 小鼠肺脏病毒复制达到高峰,为107EID50 /g,7dpi 肺脏组织病理学观察呈间质性肺炎、炎性细胞浸润; 5dpi 时IL-6 水平达到峰值,为122pg /mL; 7dpi 时IL-5 达到峰值,为680pg /mL; 与未感染组相比,感染组CD4 + CD44 +CD62L － 效应T 细胞比例显著升高。结论结果表明PＲ8 流感病毒感染小鼠,肺脏病毒复制水平高,机体免疫应答强,适合用于进一步的免疫学研究。     

[（DMPZM）HgI2]的合成与晶体结构（DMPZM=双（3，5-二甲基吡唑）甲烷）

在乙腈中,双（3,5-二甲基吡唑）甲烷（DMPZM）与HgI2反应得到化合物[（DMPZM）HgI2]（1）,该化合物通过熔点、元素分析、核磁、红外光谱等表征．X射线衍射法测得该晶体属于单斜晶系,空间群为D2 1／c,晶胞参数：a=1．2219（3）nm,b=1．4756（4）nm,c=0．9540（2）nm,β=100．309（5）°,V=1．6924（6）nm3,C11H16HgI2N4,Mr=658．67,Dc=2．585g／cm3,Z=4,F（000）=1184,R1=0．0474,ωR2=0．0991,S=0．0474．汞原子与DMPZM中的2个氮原子和2个碘原子配位,形成扭曲的四面体结构．Hg—N、Hg-I平均键长分别为0．2047nm、0．2670nm．     

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.     

H5N1的结构与致病机制

以禽流感病毒H5N1的两种基质蛋白HA、NA的结构为基础,分析了H5N1的基因结构,探索其毒力基础和致病的分子机制.     

Prediction of a common neutralizing epitope of H5N1 avian influenza virus by in silico molecular docking

The H5N1 avian influenza virus (AIV) has widely spread in Asia, Europe and Africa, making a large amount of economic loss. Recently, our research group has screened a common neutralizing mono- clonal antibody named 8H5, which can neutralize almost all H5 subtype AIV ever isolated so far. Obvi- ously, this monoclonal antibody would benefit for research and development of the universal AIV vac- cine and design of the drug against H5N1 AIV in high mutation rate. In this study, the homology mod- eling was applied to generate the 3D structure of 8H5 Fab fragment, and "canonical structure" method was used to define the specified loop conformation of CDR regions. The model was subjected to en- ergy minimization in cvff force field with Discovery module in Insight II program. The resulting model has correct stereochemistry as gauged from the Ramachandran plot calculation and good 3D-structure compatibility as assessed by interaction energy analysis, solvent accessible surface (SAS) analysis, and Profiles-3D approach. Furthermore, the 8H5 Fab model was subjected to docking with three H5 subtype hemagglutinin (HA) structures deposited in PDB (ID No: 1jsm, 2ibx and 2fk0) respectively. The result indicates that the three docked complexes share a common binding interface, but differ in bind- ing angle related with HA structure similarity between viral subtypes. In the light of the three HA inter- faces with structural homology analysis, the common neutralizing epitope on HA recognized by 8H5 consists of 9 incontinuous amino acid residues: Asp68, Asn72, Glu112, Lys113, Ile114, Pro118, Ser120, Tyr137, Tyr252 (numbered as for 1jsm sequence). The primary purpose of the present work is to provide some insight into structure and binding details of a common neutralizing epitope of H5N1 AIV, thereby aiding in the structure-based design of universal AIV vaccines and anti-virus therapeutic drugs.