编码ATR的胞外区基因cDNA的克隆、测序及表达

目的克隆并在大肠杆菌中表达编码炭疽毒素受体(anthrax toxin receptor,简称ATR)的胞外区基因。方法收集CHO-K1细胞,提取其总RNA,经反转录成单链cDNA,以此为模板PCR扩增出编码ATR胞外区基因,将该基因克隆入载体pUC19中,测序正确后,亚克隆入表达载体pMal-c2x中进行表达。结果利用所设计的引物扩增出完整的编码ATR胞外区基因。以大肠杆菌为宿主在IPTG的诱导下获得表达,激光薄层扫描显示表达蛋白占总蛋白的39%。结论获得了编码ATR胞外区基因cDNA及其原核表达产物,为进一步研究炭疽毒素致病机理和炭疽病的防治奠定了基础。     

太原市“十一五”期间科学技术杰出贡献奖

山西大学和山西省农业生物技术研究中心从我国特色资源东亚钳蝎的应用出发。采用现代生化和分子生物学研究手段，对其中的昆虫特异性毒素基因（BmKIT）和神经胶质瘤特异，眭毒素基因（BmKCT）进行克隆、定向改造，对其基因产物的性质、     

HARDY转录因子原核表达的研究

HARDY（HRD）是AP2／ERF—like家族转录因子,它的过量表达可以增强水稻的抗旱性、提高水稻的水分利用率。本研究从拟南芥中克隆了HRD转录因子基因,利用DNA重组技术,构建了HRD原核表达载体PET．HRD,并将PET－HRD重组质粒转化E．coli BL21（DE3）,经1mmol／LIPTG诱导其蛋白质表达,获得了高表达量的分子量约为42kD的HRD融合蛋白。本研究结果为进一步制备HRD转录因子的抗体,并利用该抗体来分析和克隆不同物种的HRD基因奠定了基础。     

磁电复合线材磁热效应的计算与分析

推导并分析了磁与电复合的线材（柱，管，片状）样品的磁热功率Ｐ的表达式，涡流功率可分解为与磁，电特性和样品尺寸相关，以及与磁，电复合结构（因子Ψ）和样品形状（因子η）相关的两大部分，分析导体层厚度越薄，导体对样品磁热的贡献越大，Ψ的最大值为０．５。     

一种新细胞因子基因真核表达载体的构建和鉴定

采用PCR方法,以人胎盘cDNA文库为模板,扩增出人B淋巴细胞刺激因子（hBLys）,经克隆测序及纯化后,再以此PCR产物为模板,用Nest-PCR方法进一步扩增得到B淋巴细胞刺激因子的胞膜外功能区域（hsBLyS）的DNA片段,纯化、克隆测定鉴定后,扩增并纯化粒,经酶切、纯化后克隆到真核表达质粒pcDNA3.1（＋）中,构成真核表达载体pcDNA3．1（＋）／hsBLyS。结果表明：用此方法制备得到的hsBLyS的DNA片段经测序鉴定与文献报道相符,构建的真核表达载体经鉴定也达到了预计的结果。     

期刊影响因子在评价期刊质量中的应用

影响因子(impact factor,IF)作为评价期刊质量的重要指标已为人们所熟知,总结了以影响因子为基础的评价期刊质量的指标,重点介绍了相对影响因子和论文计数影响因子。     

人肿瘤坏死因子Gln102—Glu107缺失型的构建…

基于对有关肿瘤坏死因子结构和功能关系研究资料的分析，设计了一种缺失了Ｇｌｎ １０２－Ｇｌｕ１０７位共６个氨基酸的新型肿瘤坏死因子，首先，利用ＰＣＲ技术分析扩增出Ｖａｌｌ－Ｇｙｓ１０１和Ｇｌｙ１０８－Ｌｅｕ１５７基因片段，连接后再克隆到表达载体ｐＪＬＡ５０３中，转化大肠杆菌，发酵时进行４２℃诱导表达，得到了新型肿瘤坏死因子ＣＧ－ｈＴＮＦ的产物。经过对ｐＣＧ－ｈＴＮＦ中克隆的基因进行ＤＮＡ测序鉴定，证     

Z/(n)模n剩余类环的构造

本文先讨论了Ｚ／（ｐｍ）环的结构，如其幂零元、幂等元、可逆元、零因子和理想的结构和数量．然后，利用同构知识得到了Ｚ／（ｐ１ｍ１ｐ２ｍ２…ｐｔｍｔ）环的结构，即其幂零元、幂等元、可逆元、零因子和理想的结构和数量．     

关于（1,f）—奇因子的一些新结果

设Ｇ是一个简单图，ｆ：Ｖ（Ｇ）→（１，３，５．．．．），如何对Ｇ的任意ｎ对集Ｍ，Ｇ－Ｖ（Ｍ）有一个（１，ｆ）－奇因子，则称图Ｇ存在ｎ－可扩充的（１，ｆ）－奇因子，本文主要对ｎ－可扩充图成立的一些结果进行了改进，证明了这些结果在有ｎ－可扩充的（１，ｆ）－奇因子的图中也成立。     

与任意图2-正交的(g,f)-因子分解

设G是一个图，用V(G）和E(G）表示它的顶点集和边集，并设g(x）和f(x）是定义在V(G）上的两个整数值函数，且对每个x∈V(G），有4≤g(x)≤f(x)，则图G的一个支撑子图F称为G的一个（g，f）-因子，如果对每个x∈V(G)，有g(x)≤dF(x)≤f(x)。图G的（g，f）-因子分解是指E(G）能划分成边不交的（g，f）-因子，设F={F1,F2,…,Fm}和H分别是图G的因子分解和子图，若对所有1≤i≤m有|E(H)∩E(Fi）|=2，则称F和H2-正交。本文证明：若G是一个（mg m-1，mf-m 1）-图，H是G中任一有2m条边的子图，则G有一个（g，f）-因子分解与H2-正交。     

Crystal structure of a complex between anthrax toxin and its host cell receptor

Anthrax toxin consists of the proteins protective antigen (PA), lethal factor (LF) and oedema factor (EF). The first step of toxin entry into host cells is the recognition by PA of a receptor on the surface of the target cell. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. Upon acidification of the endosome, PA63 forms a pore that inserts into the membrane and translocates EF and LF into the cytosol. Two closely related host cell receptors, TEM8 and CMG2, have been identified. Both bind to PA with high affinity and are capable of mediating toxicity. Here, we report the crystal structure of the PA-CMG2 complex at 2.5 A resolution. The structure reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the extracellular matrix by integrins. The binding surface is closely conserved in the two receptors and across species, but is quite different in the integrin domains, explaining the specificity of the interaction. CMG2 engages two domains of PA, and modelling of the receptor-bound PA63 heptamer suggests that the receptor acts as a pH-sensitive brace to ensure accurate and timely membrane insertion. The structure provides new leads for the discovery of anthrax anti-toxins, and should aid the design of cancer therapeutics.     

Anthrax toxins cooperatively inhibit endocytic recycling by the Rab11/Sec15 exocyst

Bacillus anthracis is the causative agent of anthrax in humans and other mammals. In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and oedema factor (EF), leading to widespread vascular leakage and shock. Whereas host targets of LF (mitogen-activated protein-kinase kinases) and EF (cAMP-dependent processes) have been implicated in the initial phase of anthrax, less is understood about toxin action during the final stage of infection. Here we use Drosophila melanogaster to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF. EF reduces levels of apically localized Rab11 and indirectly blocks vesicle formation by its binding partner and effector Sec15 (Sec15-GFP), whereas LF acts more directly to reduce Sec15-GFP vesicles. Convergent effects of EF and LF on Rab11/Sec15 inhibit expression of and signalling by the Notch ligand Delta and reduce DE-cadherin levels at adherens junctions. In human endothelial cells, the two toxins act in a conserved fashion to block formation of Sec15 vesicles, inhibit Notch signalling, and reduce cadherin expression at adherens junctions. This coordinated disruption of the Rab11/Sec15 exocyst by anthrax toxins may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.     

Identification of the cellular receptor for anthrax toxin.

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.     

Crystal structure of the anthrax lethal factor.

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.     

Screening inhibitors of anthrax lethal factor

The disease anthrax is caused by lethal factor, an enzyme component of the toxin produced by the spore-forming bacterium Bacillus anthracis. Here we describe substrate molecules for this factor that offer a means for high-throughput screening of potential inhibitors for use in anthrax treatment. Our assay should help to answer the urgent call for new and specific therapies to combat this pathogen after its recent emergence as a terrorist bioweapon.     

Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin

Anthrax poses a clear and present danger as an agent of biological terrorism. Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death. Anthrax lethal toxin (LT) is a critical virulence factor of B. anthracis, which occurs as a complex of protective antigen and lethal factor. Here we demonstrate that LT severely impairs the function of dendritic cells--which are pivotal to the establishment of immunity against pathogens--and host immune responses by disrupting the mitogen-activated protein (MAP) kinase intracellular signalling network. Dendritic cells exposed to LT and then stimulated with lipopolysaccharide do not upregulate co-stimulatory molecules, secrete greatly diminished amounts of proinflammatory cytokines, and do not effectively stimulate antigen-specific T cells in vivo. Furthermore, injections of LT induce a profound impairment of antigen-specific T- and B-cell immunity. These data suggest a role for LT in suppressing host immunity during B. anthracis infections, and represent an immune evasion strategy, where a microbe targets MAP kinases in dendritic cells to disarm the immune response.     

一种功能高分子支载化合物对沙林的滤毒作用研究

本文报导一种模拟乙酰胆碱脂酶的某些功能的高分子支载化合物:对神经毒剂沙林的滤毒防毒作用。试验表明,其防滤时间是“通用”滤毒活性碳4号的3倍,再生后的功能高分子支载化合物的滤防时间变化不大。     

The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4

Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens. Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns--including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA--and in turn activate effector functions, including anti-apoptotic signalling pathways. Certain pathogens, however, such as Salmonella spp., Shigellae spp. and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis. We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase. We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4. Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis. The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.     

毒隐翅虫研究进展：2.毒素及其在医学上的重要意义

本文对毒隐翅虫毒素的分析、合成、生化特性,在虫体内的出处,毒理学与病理学,药理学,在癌及细胞生物学上的意义作了综述.     

理化因素对As—3毒素活性的影响研究

报道了温度，ｐＨ，蒸馏水，酶等环境因素对Ａｓ－３毒素活性的影响，为Ａｓ－３毒素的进一步研究及高效应用可提供有意义的参数。