Screening and Identification of Mimotopes of Highly Pathogenic H5N1 Avian Influenza A Virus HA

摘要 禽流感（Avianinfluenza,AI）是由A型流感病毒引起的烈性传染病。全球自2003年以来，已有10个国家先后报告发现人感染H5N1病例，导致170多名患者死亡，人类防控行动不可懈怠。在禽流感病毒编码的各种蛋白中，以血凝素（HA）基因的突变率最高。而且HA是流感病毒最主要的表面抗原，它能够诱导机体产生相应的中和抗体。本实验室制备的H5N1抗体库中，已发现有4株抗体对目前所检测的33株不同遗传变异亚系的H5N1病毒代表株均能有效中和，提示H5N1病毒HA上可能存在重要的高保守性中和位点。本实验利用其中的1个保守中和单抗8H5，应用噬菌体展示技术筛选HA中和表位模拟肽，为禽流感...Abstract Avian Influenza (AI) is one of syndrome disease of birds caused by influenza A virus in avian. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health (OIE). The disease broke out in many countries of the world, and resulted in a serious economic loss. At the same time, it threatens the public health. The HA is th...学位：理学硕士院系专业：生命科学学院生物化学与生物技术系_生物化学与分子生物学学号：20042608

The Study Progress of Alterative Oxidase in Higher Plant

交替氧化酶(Alternative Oxidase,AOX)广泛存在于高等植物、藻类和原生生物线粒体内膜。从主呼吸链的辅酶Q分岔,是氧化辅酶Q、还原氧分子生成水的另一终端氧化酶。氧化过程没有质子穿膜运动、热量以产热方式散发。产热植物中交替氧化产生的热量使花粉发出芳香味吸引虫传粉。推测植物AOX使植物在环境胁迫下维持呼吸,调节能量平衡,抵抗氧化胁迫,保持三羧酸循环的运行。AOX是首次发现的双铁羧酸蛋白质成员中的膜蛋白质,AOX与膜分离后容易失活,至今尚未有三级结构的报导,只有二级结构的2种假设模式,最新的模式AOX为膜界面蛋白质而不是跨膜蛋白。最近我们的研究表明有2个途径可获得适量有活性的AOX:建立优化的pFLAG-1-AOX大肠杆菌超量表达系统;从产热植物如斑叶阿若母(Arum maculatum)花序组织线粒体分离纯化有活性的AOX。Alternative oxidase(AOX),a terminal oxidase located in the inner mitochondrial membrane,has been identified universally in plant,algae,fungi,and protozoan.AOX branches from the cytochrome pathway at the level of the ubiquinone and catalyses the oxidation of ubiquinol and reduction of oxygen to water.There is non-protonmotive and energy is liberated as heat instead of producing ATP.In thermogenic plants,heat released by AOX attracts insects for pollination by heating aromatic compounds.The use of AOX in plant may(a) allow flexibility of respiration under changing environmental conditions(b) regulate energy homeostasis(c) help prevent oxidative stress(d) maintain TCA cycle turnover.AOX is a member of di-iron carboxylate proteins.Two structural models exist for AOX and the latest one is a monotopic protein instead of span membrane.Relatively little is known about its 3-dimensional structure.Main reason is that the sufficient quantities of AOX suitable for crystallogram structural studies is unavailable because of its unstableness.Recently we tried to obtain sufficient quantities alternative oxidase protein(AOX) for further construction studies.The aim is achieved by over-expressing in E.coli and improving purification of native AOX from Arum mitochondrial.国家自然科学基金(No30570104

A Theoretical Study of the Mechanism for Allylic Ether Isomerization

采用MP2和密度泛函M06-2X方法,在6-31++g(d,P)基组水平上对烯丙基类不对称醚异构化反应机理进行了计算研究.揭示了其可能的反应途径,预测了互变异构吉布斯自由能,活化能等性质.计算结果表明,在没有金催化剂的条件下,尽管有醇溶剂时异构化活化能垒有所降低,异构化反应依然不容易进行.相反,存在金催化剂并且有醇溶剂情况下,烯丙基类不对称醚异构化反应活化自由能大大降低,仅为7.5 kCAl/MOl.通过比较有无醇溶剂和金催化剂对异构化的影响,揭示了金烯烃络合和醇分子参与反应以质子转移的异构化反应机理,很好解释了实验中观察的现象.计算结果还表明:醇分子不仅参与反应提供质子转移,它还能与醚竞争金催化剂络合,因此在高浓度醇条件下会抑制异构化反应进行.The reaction mechanism of allylic ether isomerization has been investigated by MP2 and DFT method(different functionals) with 6-31++G(d,p) basis set.The calculated results show that M06-2X method that designed to treat dispersion and hydrogen-bonded systems do better than traditional functional-B3LYP for the calculated energetic and structural properties of allylic ether isomerization.The optimal structures of allylic ether and transition states were located and the reaction Gibbs free energy barriers were predicted at the MP2 and M06-2X level.Furthermore, the possible reaction pathways and mechanisms were proposed to explain the origin of regioselectivity observed in experiment.The calculation results show that the isomerization reaction will not readily occur in the absence of catalysis by Au.The computed potential energy barrier is quite high, and things get better when alcohol molecules are introduced, resulting in the decrease of calculated activation free energy from 67.1 to 48.6 kcal/mol.However, the Au(I)-catalyzed addition of another molecule of alcohol to an allylic ether can occur readily.A protonated diether intermediate was stabilized by a hydrogen bond and the activation energies of allylic ether isomerization were dramatically decreased, only 7.5 kcal/mol.By contrast the isomerization effect under with and without alcohol, gold catalysis, the results indicate that the allylic ether isomerization involve cationic gold coordination and proton shift reaction process, which form the intermediate that allows the interconversion of the products.This reaction mechanism can successfully explain the observed regioselectivity for the thermodynamic product.Meanwhile, the results also show that the isomerization was completely inhibited with the excess alcohol due to competing gold coordination between alcohol and ether.The discovery of gold catalysts in allylic ether isomerization not only contributes to the development of catalysts from the usual transition metals to noble metals, but also shows the potential catalytic activities by switching the reaction conditions.国家自然科学基金(No.21273177); 西部之光计划资助~

In vitro Micro-root Tuber Formation of Pseadostellaria heterophylla Miq.and Preliminary Study of Its Application

太子参组培苗单茎节段插入含45g·l-1蔗糖的MS培养基中,于60μMOl·M-2·S-1光照强度下培养120d后,其节部位长出不定根膨大形成与太子参体内块根相似、重量约为块根1/20的微块根,平均每株可形成1.54个微块根。微块根移植入大田后,其生长特性和物候期与大田块根相似,都有低温萌芽、高温休眠的特性;开花结果、不定根膨大、块根成熟等也相似。同时,组培中的微块根还具有易培养、耐贮存、体积小、易移栽和繁殖系数高等优点,在生产中具有很大的应用潜力。When sterile stems of 'Taizishen'(Pseadostellaria heterophylla) were put in MS medium with 45 g·L-1 sucrose in 60 μmol·m-2·s-1 illumination for 120 days,adventitious roots from nodes would enlarge and form micro-root tubers which were same with root tubers in vivo,but had a 1/20 weight of the latter.Each plantlet averagely produced 1.54 micro-root tubers.When the micro-root tubers plateded in field,they displayed same growth features and phenophase with root tubers in vivo,such as germination in low temperature and dormancy in higher temperature.The plants from micro-root tubers also could flower and set up seeds,and its adventitia roots could enlarge and form mature root tubers.The micro-root tubers were small in size,easy of culture and transplant,convenience of storage and higher ability of propagation,which had great potential application in production of P.heterophylla.厦门市科技计划(3502Z20084037

Ultra-structural Changes in Pollen Development of Lycium barbarum L.

用JEM-100CXⅡ透射电子显微镜对宁夏枸杞(Ly cium barbarum L.)花粉发育过程做了超微结构观察.结果表明,在花粉母细胞细线和偶线期,核糖体数量减少、线粒体结构简化;之后核糖体数量逐渐回升,后期Ⅰ线粒体结构恢复正常.小孢子液泡化过程中核糖体再次减少,同时线粒体和质体结构简化;在早期二胞花粉中,核糖体数量增加、线粒体和质体结构再度分化.花粉母细胞和小孢子发育过程中都存在“细胞质改组”现象,且这两次“细胞质改组”与细胞功能的转变密切相关.在次生造孢细胞、花粉母细胞和小孢子后期都有液泡数量明显增加或体积增大的过程.前两次液泡的增加可能参与了其胼胝质壁的构建;而小孢子晚期中的大液泡则创造了一种极性为其不等分裂作好了生理和结构上的准备.The ultra-structural observation of Lycium barbarum L.pollens was conducted by JEM-100CXⅡ transmission electron microscope.The results showed that the mother pollen cells presented decreased ribosome numbers at leptotene and zygotene stages,and simplified mitochondria structure;afterwards,their ribosomes gradually increased in quantity and their mitochondria recovered their normal structures at prophase Ⅰ.During microspore vacuolization,their ribosomes declined in quantity again and their mitochondria and plastids became simplified in their structures;in bicellular pollens at the early stage the ribosomes increased in quantity and the mitochondria and plastids differentiated in their structure once again.In the developments of both the mother pollen cells and microspores,there existed the phenomenon of "cytoplasm reorganization" and the two "cytoplasm reorganization" closely related to the transformation of cellular functions.At the late stage of the sporogenous cells,mother pollen cells and microspores,the vacuoles remarkably increased in quantity or size.The first two vacuole increases probably involved callose-wall construction;big vacuoles in the microspore at the advanced stage created a polarity to prepare themselves physiologically and structurally ready for unequal division.国家自然科学基金项目(30570104

Micro-Root Tuber Development and Histochemistry Study of Pseudostellaria heterophylla Miq.

采用植物组织培养、解剖学及组织化学定位方法研究太子参试管微块根发育的形态结构与营养物质积累特征的结果表明:太子参微块根由组培苗膨大的腋芽基部长出的不定根发育而成,经历了初生结构与次生结构发育,其膨大加粗是由于不定根的次生生长。维管形成层向内形成大量的次生木质部构成微块根的主要部分。淀粉粒是太子参微块根的主要营养存储方式。随着微块根的次生生长,淀粉粒先在次生木质部薄壁细胞中形成,随后在次生韧皮薄壁细胞中也大量积累。膨大的微块根可以合成太子参皂苷,成熟微块根中次生韧皮部的皂苷含量略高于次生木质部。离体太子参微块根的生长发育和营养物质的积累与块根中的相同。The feature of morphology and structure of in vitro micro-root tubes of Pseudostellaria heterophylla Miq.and their nutrition accumulation were studied using anatomy and histochemistry methods.The micro-root tubers came from inflated adventitious roots of axillary bud.The secondary growth of micro-root tubers made them enlarge.Its vascular cambium formed a large number of xylem parenchyma cells which consisted of main structure of the tuber.The micro-root tubers synthesized starches during its enlargement.Starches were the mainly storage material in the micro-root tubers,which were stored in xylem parenchyma cells.With the secondary growth of micro-root tubers,starches were firstly formed in secondary xylem parenchyma cells,and then abundant starches were also accumulated in most secondary phloem parenchyma cells.In later microroot tubers,the xylem and phloem parenchyma cells synthesized saponin materials which could be detected using a histochemical method.The development and nutrition accumulation of in vitro micro-root tubers of P.heterophylla are as same as in vivo,which will provide an assay system of P.heterophylla to investigate its tuber development and application.厦门市科技计划项目(3502Z20084037);宁德市科技计划项目(20090024

Optimal Expression of Alternative Oxidase in Escherichia coli

植物交替氧化酶(Alternative Oxidase,AOX)位于高等植物线粒体内膜,从细胞色素途径的辅酶Q分岔,催化4个电子还原氧分子形成水的另一终端氧化酶。分离纯化有活性的AOX比较困难。本文研究AOX原核表达,选择pFLAG-1分泌表达载体,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导AOX优化表达,pFLAG-1-AOX大肠杆菌优化表达条件为:宿主DH5α、温度37℃、细胞密度OD600=0.6、IPTG浓度0.2mmol/L,诱导后60min收获细胞;获得少量可溶的细胞外周质AOX和大量不溶的AOX,为深入研究AOX打下基础,同时为研究膜蛋白原核表达提供依据。Alternative oxidase(AOX),a terminal oxidase located in the inner mitochondrial membrane,has been identified universally in plant,algae,fungi and protozoan.AOX branches from the cytochrome pathway at the level of the ubiquinone and catalyses the oxidation of ubiquinol and reduction of oxygen to water.The enzyme remains difficult to purify to homogeneity in a stable,active form.The paper expressed the AOX with inducer IPTG in pFLAG-1 Escherichia coli system.pFLAG-1 carry a ompA which secretary the AOX to periplasm.Three strategies have combined to increase the yield of AOX.First strategy involved tuning of the inducer concentration while the second involved alter the growth temperature and the last involved differ the cell concentration.The optimized protocol is that the host cell is DH5α,the temperature is 37℃,the cell concentration is OD600=0.6 and the IPTG is 0.2 mmol/L.Purification of proteins showed AOX is expressed in unsoluble protein.国家自然科学基金(30570104

噬菌体抗体库固相筛选条件的初步研究

目的:探讨噬菌体抗体库的固相筛选条件,为筛选方案的设计提供实验依据。方法:利用多种针对HEVNE2蛋白的特异性噬菌体人源抗体和非特异性噬菌体人源抗体,对噬菌体抗体与抗原的结合时间、抗原包被的浓度、洗涤强度和洗脱方式等多种筛选的条件进行初步探索。结果:阳性噬菌体抗体与抗原反应1min,就可较好结合,洗涤次数为20～30次、洗涤液的pH为5时,筛选得到的阳性率最高。包被抗原的浓度对筛选的阳性率没有明显影响,用10mg/L抗原竞争洗脱60min,可得到较高的阳性率。结论:噬菌体抗体库的筛选是一个非常复杂的过程,其中的各个条件之间有着密切的联系,应该根据具体情况进行调整

多样性人源天然噬菌体抗体库的构建及初步应用

目的:构建多样性良好的人源天然噬菌体抗体库。方法:从正常人外周血中分离淋巴细胞,以RT-PCR和半巢式PCR扩增重链可变区VH基因和轻链可变区VL基因,以重叠延伸PCR将VH、VL组装成scFv基因,并将其克隆入噬菌粒载体pCANTAB-5E中。以pCANTAB-5E电转化大肠杆菌TG1,构建人源天然噬菌体抗体库,测序分析抗体基因的家族信息和多样性,并用多种抗原对其进行筛选。结果:获得了库容为2×108的人源天然噬菌体抗体库。分别用5种抗原对其进行筛选,均可获得特异性噬菌体抗体的富集。结论:成功地构建了一个多样性良好的人源天然噬菌体抗体库,可用于制备具有应用前景的人源抗体

亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体