Construction of eukaryotic vector pcDNA3.1-flag-pygo2 and expression in C6 cell

目的构建pcDNA3.1-flag-pygo2真核表达载体并在C6细胞中进行表达。方法从小鼠脑胶质细胞中提取总RNA,RT-PCR法反转录合成c DNA,设计引物,调取目的片段,与pcDNA3.1-flag载体连接后转化大肠杆菌DH5α,LB平板筛选菌落,提取质粒。重组质粒pcDNA 3.1-flag-pygo2经过酶切鉴定及测序后,阳离子脂质体法转染C6细胞并经免疫细胞化学染色及蛋白印迹检测重组体的表达。结果重构质粒pcDNA 3.1-flag-pygo2经限制性核酸内切酶EcoRⅠ,HindⅢ酶切分析及测序检查,表明真核表达载体构建正确;瞬时转染C6细胞后,免疫细胞荧光染色及蛋白印迹检测表明转染细胞能够表达外源Pygo2基因。结论成功构建了pcDNA3.1-flag-pygo2真核表达载体并能够在真核细胞中进行表达,这为今后研究pygo2基因在胶质瘤中的作用机制奠定了基础。Objective To construct the eukaryotic expression vector pcDNA3.1-flag-pygo2 and express the combined protein in C6 cell line.Methods To extract total RNA from primary glial cell of mouse and to synthesize c DNA by RT-PCR,then design primer and clone whole segment of pygo2 gene.After the targeted gene was inserted into vector pcDNA3.1-flag,the recombined plamid was transformed into E coli DH5α for LB agar plate screening and the recombined plasmid were extracted and purified.After verification by double enzyme digestion and sequencing.,the constructed eukaryotic expression plamid was transfected to C6 cell line by lipofectamin method,finally the protein expression was detected by immunocytochemical staining as well as western blot.Results The new constructed vector was confirmed by restricted enzyme Eco R I,HindIII digestion assay and correct Pygo2 was verified by sequenceing.finally,pcDNA3.1-flag-hpygo2 can express exogenous pygo2 gene in glioma C6 cell line after transient transfection by the determination of immunocytofluorescent staining and western blot.Conclusion The new plamid pcDNA3.1-flag-pygo2 was constructed successfully,and can express fused protein in eukaryotic cell,which establish the foundation for future research on pygo2 gene function in human glioma

Application of Fluorescent Probe Technique in Study of Polymer Self-Assembly

采用自组装技术制备新型功能性材料已成为聚合物材料的一个热门研究领域。大部分两亲性聚合物,如嵌段聚合物、接枝聚合物、星型聚合物、树枝状聚合物和部分无规聚合物及聚电解质,在特定条件下可发生自组装。在聚合物自组装的研究中,荧光技术已经得到了广泛的应用,尤其是荧光探针技术。根据荧光探针分子荧光光谱特征峰荧光波长、强度、偏振以及寿命等参数的变化,可以简便而又准确地研究聚合物的临界胶束浓度、温度和PH敏感性、结构与自组装形态的关系、微环境变化等信息。本文综述了荧光探针技术在两亲性聚合物自组装行为研究中的应用,重点介绍了荧光探针技术研究聚合物亲水亲油平衡值(Hlb值)、浓度、温度、PH、溶剂组成及离子强度等因素对聚合物自组装形貌和微观特性参数的影响。此外,结合我们的研究工作对本征荧光光谱方法在聚合物研究中的应用做了阐述和展望,以期为两亲性聚合物的设计合成、自组装行为控制及应用提供参考。Preparation of new functional materials by self-assembly technique has become a hot research field of polymeric materials science.Most of the amphiphilic polymers,such as block copolymers,graft copolymers,star copolymers,dendrimers,part of the random copolymer and polyelectrolyte,etc.can self-assemble under certain conditions.In the study of polymer self-assembly,fluorescent technique has been widely used,especially fluorescent probe technique.According to the changes of the characteristic fluorescence parameters of the probe molecules,such as wavelength,intensity,polarization,lifetime,etc.the critical micelle concentration,temperature and pH dependence,the relationship of structure and self-assemble morphology of the polymer could be easily and accurately studied.This paper is focused on the application of the fluorescent probe technique in investigation of self-assembly behavior of amphiphilic polymers.The effects of hydrophilic lipophilic balance(HLB),concentration,temperature,pH,solvent composition,ionic strength,etc.on self-assemble morphology and microscopic characteristics parameters of amphiphilic polymers are particularly reviewed.Furthermore,based on our own research work,the applications of intrinsic fluorescence spectroscopy method in polymer investigation are elaborated.It will characterize the conformation transitions of macromolecules during the self-assembly process more truly.This paper aims at providing reference for design,polymerization,self-assembly controlling and applications of amphiphilic polymers.国家自然科学基金项目(No.51103123); 福建省自然科学基金项目(No.2012J01234)资

南海北部天然气水合物的形成分解与微生物的偶联关系

微生物在天然气水合物的形成和分解中扮演了重要的角色.南海北部是我国天然气水合物未来开发的战略选区之一,目前已多次在该海域采集到天然气水合物样品,证实了南海北部蕴藏着丰富的天然气水合物资源.通过分析天然气水合物形成与分解同微生物的偶联关系,综述了与天然气水合物形成分解有关的微生物类型及其标志化合物,结合我国南海北部天然气水合物赋存或潜在赋存区的微生物相关研究工作进展,提出未来使用微生物地球化学方法勘探天然气水合物的技术指标和相关的研究方向.国家自然科学基金(41773078,41276046);;厦门大学校长基金(0050-ZK1104

Atomic structures of enterovirus D68 in complex with two monoclonal antibodies define distinct mechanisms of viral neutralization

11月5日，《自然》子刊《自然•微生物学》（Nature Microbiology）在线刊出了我校夏宁邵教授团队发表的题为“Atomic Structures of Enterovirus D68 in Complex with Two Monoclonal Antibodies Define Distinct Mechanisms of Viral Neutralization”的研究论文。这是夏宁邵教授团队在《自然•通讯》（Nature Communications，2017）、《科学•进展》（Science Advances，2018）上发表手足口病重要病原体CVA6、CVA10研究论文之后的又一项关于肠道病毒的重要研究成果。该研究通过解析肠道病毒D组68型（EV-D68）不同类型病毒颗粒及其免疫复合物的高分辨率结构，系统阐明了EV-D68病毒的生活周期及各时期的病毒中和机制，进一步完善了小RNA病毒的吸附入胞及感染机制理论，为EV-D68新型疫苗、抗病毒治疗药物的研发提供重要的理论指导。该研究依托电镜技术平台，解析了EV-D68病毒生活周期中的三种代表性颗粒成熟颗粒、脱衣壳中间态和前体病毒衣壳的近原子分辨率结构，阐明了三种病毒颗粒间的结构差异，以及成熟颗粒转变为脱衣壳中间态的分子机制。夏宁邵教授、李少伟教授、程通副教授和美国国立卫生研究院（NIH）高级研究员Barney Graham博士为该论文的共同通讯作者。郑清炳工程师、博士生朱瑞、博士后徐龙发、博士生何茂洲和美国加州大学圣地亚哥分校颜晓东博士为该论文共同第一作者。【Abstract】Enterovirus D68 (EV-D68) undergoes structural transformation between mature, cell-entry intermediate (A-particle) and empty forms throughout its life cycle. Structural information for the various forms and antibody-bound capsids will facilitate the development of effective vaccines and therapeutics against EV-D68 infection, which causes childhood respiratory and paralytic diseases worldwide. Here, we report the structures of three EV-D68 capsid states representing the virus at major phases. We further describe two original monoclonal antibodies (15C5 and 11G1) with distinct structurally defined mechanisms for virus neutralization. 15C5 and 11G1 engage the capsid loci at icosahedral three-fold and five-fold axes, respectively. To block viral attachment, 15C5 binds three forms of capsids, and triggers mature virions to transform into A-particles, mimicking engagement by the functional receptor ICAM-5, whereas 11G1 exclusively recognizes the A-particle. Our data provide a structural and molecular explanation for the transition of picornavirus capsid conformations and demonstrate distinct mechanisms for antibody-mediated neutralization.This work was supported by a grant from the National Science and Technology Major Projects for Major New Drugs Innovation and Development (no. 2018ZX09711003-005-003), the National Science and Technology Major Project of Infectious Diseases (no. 2017ZX10304402-002-003), the National Natural Science Foundation of China (no. 81401669 and 81801646) and the Natural Science Foundation of Fujian Province (no. 2015J05073). This work was supported in part by funding by the National Institutes of Health (grants R37-GM33050, GM071940, DE025567 and AI094386). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and by instrumentation grants from the NIH (1S10RR23057 and 1U24GM116792) and NSF (DBI-1338135 and DMR-1548924). 该研究获得了国家自然科学基金、新药创制国家科技重大专项、传染病防治国家科技重大专项和美国国立卫生研究院基金的资助

Discovery and structural characterization of a therapeutic antibody against coxsackievirus A10

9月20日，《科学》子刊《科学•进展》（Science Advances）刊出了我校夏宁邵教授团队发表的题为“Discovery and structural characterization of a therapeutic antibody against coxsackievirus A10”的研究论文。该研究首次发现手足口病重要病原体柯萨奇病毒A组10型（CVA10）不同类型病毒颗粒共有的优势中和表位，揭示了病毒颗粒及其与优势中和抗体复合物的精确三维结构，阐明了中和抗体的功能与作用机制，为新型疫苗和治疗药物的研制提供了重要的理论基础。 该研究首次揭示并描绘了CVA10的病毒颗粒及其优势中和表位的精确特征，发现了具有良好应用潜能的治疗性中和抗体，为新型疫苗和特异性治疗药物的研究提供了关键基础。 我校夏宁邵教授、程通副教授和美国加州大学洛杉矶分校纳米系统研究所Z. Hong Zhou（周正洪）教授、美国加州大学圣地亚哥分校颜晓东博士为该论文的共同通讯作者。我校博士生朱瑞、徐龙发博士后、郑清炳工程师、李少伟教授和美国加州大学洛杉矶分校崔彦祥博士后为该论文共同第一作者。【Abstract】Coxsackievirus A10 (CVA10) recently emerged as a major pathogen of hand, foot, and mouth disease and herpangina in children worldwide, and lack of a vaccine or a cure against CVA10 infections has made therapeutic antibody identification a public health priority. By targeting a local isolate, CVA10-FJ-01, we obtained a potent antibody, 2G8, against all three capsid forms of CVA10. We show that 2G8 exhibited both 100% preventive and 100% therapeutic efficacy against CVA10 infection in mice. Comparisons of the near-atomic cryo–electron microscopy structures of the three forms of CVA10 capsid and their complexes with 2G8 Fab reveal that a single Fab binds a border region across the three capsid proteins (VP1 to VP3) and explain 2G8’s remarkable cross-reactivities against all three capsid forms. The atomic structures of this first neutralizing antibody of CVA10 should inform strategies for designing vaccines and therapeutics against CVA10 infections.This work was supported by grants from the National Science and Technology Major Projects for Major New Drugs Innovation and Development (2018ZX09711003-005-003), the National Science and Technology Major Project of Infectious Diseases (2017ZX10304402-002-003), the National Natural Science Foundation of China (31670933 and 81801646), and the National Institutes of Health (R37-GM33050, GM071940, DE025567, and AI094386). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by the University of California, Los Angeles and by instrumentation grants from NIH (1S10RR23057 and 1U24GM116792) and NSF (DBI-1338135 and DMR-1548924). 该研究获得了国家自然科学基金、新药创制国家科技重大专项、传染病防治国家科技重大专项和美国国立卫生研究院基金的资助

Atomic structures of Coxsackievirus A6 and its complex with a neutralizing antibody

手足口病是一种由人肠道病毒引起的全球性传染病，主要发生于5岁以下的婴幼儿，严重危害公众健康。根据获得的手足口病流行病学和病原学调查数据，目前认为CVA6与EV71和CVA16一样应作为优先的手足口病疫苗预防对象，亟需研制有效的预防和治疗方法。然而令人遗憾的是，目前对于CVA6的基础病毒学特别是结构生物学知识均缺乏足够了解，严重制约了相关研究的有效开展。 夏宁邵教授团队研究首次揭示了手足口病重要病原体柯萨奇病毒A组6型（CVA6）的病毒颗粒及其与中和抗体复合物的精确三维结构，为新型疫苗和治疗药物的研制提供了重要的理论基础。这项研究发现并精确描绘了CVA6的病毒颗粒及其与优势中和抗体的结构特征，首次完成了对CVA6的高精度“成像”，为新型疫苗和治疗药物研制提供了关键基础。 该研究工作在厦门大学分子疫苗学和分子诊断学国家重点实验室、国家传染病诊断试剂与疫苗工程技术研究中心科研平台完成。夏宁邵教授、颜晓东博士、程通副教授为该研究论文的共同通讯作者。颜晓东博士来自美国加州大学圣地亚哥分校，同时受聘为我校双聘教授。共同第一作者为徐龙发博士生、郑清炳工程师和李少伟教授。【Abstract】Coxsackievirus A6 (CVA6) has recently emerged as a major cause of hand, foot and mouth disease in children worldwide but no vaccine is available against CVA6 infections. Here, we demonstrate the isolation of two forms of stable CVA6 particles-procapsid and A-particle-with excellent biochemical stability and natural antigenicity to serve as vaccine candidates. Despite the presence (in A-particle) or absence (in procapsid) of capsid-RNA interactions, the two CVA6 particles have essentially identical atomic capsid structures resembling the uncoating intermediates of other enteroviruses. Our near-atomic resolution structure of CVA6 A-particle complexed with a neutralizing antibody maps an immune-dominant neutralizing epitope to the surface loops of VP1. The structure-guided cell-based inhibition studies further demonstrate that these loops could serve as excellent targets for designing anti-CVA6 vaccines.This work was supported by a grant from the National Natural Science Foundation of China (No. 31670933 and 81401669), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2017ZX09101005-005-003), the National Science and Technology Major Project of Infectious Diseases (No. 2017ZX10304402-002-003) and the Natural Science Foundation of Fujian Province (No. 2015J05073). This work was also supported in part by funding to T.S.B. from the National Institutes of Health (Grant R37-GM33050). 研究工作也得到了国际病毒结构生物学权威专家美国加州大学洛杉矶分校周正洪教授的大力支持和帮助，获得了国家自然科学基金、新药创制国家科技重大专项、传染病防治国家科技重大专项和福建省自然科学基金的资助

基于布里渊散射特性的光纤传感系统的设计

文章在深入分析各种光纤物理特性的基础上,根据不同的应用环境对传感光纤做了选择,搭建了基于全光纤Mach-Ze-hnder干涉仪的布里渊散射谱测量系统,实现了对所选传感光纤在常温下布里渊频移的定性测量,并对普通通信单模光纤的布里渊频移的温度依赖性进行了定性测量。设计了基于布里渊散射的光时域反射计

不同倍性葡萄种子萌芽力研究

以二倍体酿酒葡萄品种‘北红’‘北玫’与其同源四倍体‘四倍体北红’‘四倍体北玫’的种子为试材,研究其在恒温条件(25℃,24 h)下与变温条件(30℃,12 h/20℃,12 h)下萌芽力的差异。结果表明:两种萌发环境处理下二倍体‘北红’‘北玫’种子的萌芽率均明显高于相应的同源四倍体种子,二倍体种子的萌发速率也快于其同源四倍体。无论是变温处理还是恒温处理,对二倍体和其同源四倍体种子的最终萌发率均没有明显影响,但变温处理降低了种子的萌芽速率,延长了萌发所需时间,同时使萌发更为集中

research on quick response of apparel supply chain for collaboration

我国服装企业存在库存高和客户满足率低的矛盾,解决这对矛盾的一个有效方法是建立面向协同的供应链快速反应机制。通过分析这对矛盾的根本原因,提出了面向协同的服装供应链快速反应机制的解决方案,并对其中主要相关的服装设计开发、物流分销和信息化等策略与方法进行了阐述

Design of Memory Alloy Wire Connector for Pressure Releasing Device

为实现用于基于记忆合金的回转型压紧释放装置中的记忆合金与驱动元件的连接,设计出一种互换性强的连接机构,以实现不需要更换连接件或拆卸驱动元件,就可安装不同直径的记忆合金丝。通过介绍并选择记忆合金的驱动方式,分析连接指标,设计出连接机构中的凸轮和夹座,并对销进行理论校核,最后根据某压紧释放装置的需要为例,分析了夹座的强度,校核销的可靠性,验证机构的合理性